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The α3(IV)185–206 Peptide from Noncollagenous Domain 1 of Type IV Collagen Interacts with a Novel Binding Site on the β3Subunit of Integrin αvβ3 and Stimulates Focal Adhesion Kinase and Phosphatidylinositol 3-Kinase Phosphorylation*

  • Sylvie Pasco
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  • Jean-Claude Monboisse
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  • Nelly Kieffer
    Correspondence
    To whom correspondence should be addressed: Laboratoire Franco-Luxembourgeois de Recherche Biomédicale, Centre Universitaire, 162A avenue de la Faı̈encerie, L-1511 Luxembourg, Luxembourg. Tel.: 352-466644-440; Fax: 352-466644-442
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  • Author Footnotes
    * This work was supported by grants from the Center de Recherche Public-Santé, Luxembourg; CNRS, France; the Fondation Luxembourgeoise contre le Cancer and EC Biomed Grant BMH4-CT98-3517.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.
    § Recipient of a postdoctoral fellowship from the Association pour la Recherche sur le Cancer, France.
Open AccessPublished:October 20, 2000DOI:https://doi.org/10.1074/jbc.M005235200
      We have recently identified integrin αvβ3 and the associated CD47/integrin-associated protein (IAP) together with three other proteins as the potential tumor cell receptors for the α3 chain of basement membrane type IV collagen (Shahan, T.A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584–4590). Using different cell lines expressing αvβ3, αIIbβ3, and/or CD47 and a liquid phase receptor capture assay, we now provide direct evidence that the synthetic and biologically active α3(IV)185–206 peptide, derived from the α3(IV) chain, interacts with the β3 subunit of integrin αvβ3, independently of CD47. Increased α3(IV) peptide binding was observed on transforming growth factor-β1-stimulated HT-144 cells shown to up-regulate αvβ3 independently of CD47. Also, incubation of HT-144 melanoma cells in suspension induced de novo exposure of ligand-induced binding site epitopes on the β3 subunit similar to those observed following Arg-Gly-Asp-Ser (RGDS) stimulation. However, RGDS did not prevent HT-144 cell attachment and spreading on the α3(IV) peptide, suggesting that the α3(IV) binding domain on the β3subunit is distinct from the RGD recognition site. α3(IV) peptide binding to HT-144 cells in suspension stimulated time-dependent tyrosine phosphorylation, while the RGDS peptide did not. Two major phosphotyrosine proteins of 120–130 and 85 kDa were immunologically identified as focal adhesion kinase and phosphatidylinositol 3-kinase (PI3-kinase). A direct involvement of PI3-kinase in α3(IV)-dependent β3 integrin signaling could be documented, since pretreatment of HT-144 cells with wortmannin, a PI3-kinase inhibitor, reverted the known inhibitory effect of α3(IV) on HT-144 cell proliferation as well as membrane type 1-matrix metalloproteinase gene expression. These results provide evidence that the α3(IV)185–206 peptide, by directly interacting with the β3 subunit of αvβ3, activates a signaling cascade involving focal adhesion kinase and PI3-kinase.
      FAK
      focal adhesion kinase
      CHO
      Chinese hamster ovary
      LIBS
      ligand-induced binding site
      mAb
      monoclonal antibody
      MMP
      matrix metalloproteinase
      MT1-MMP
      membrane type 1-matrix metalloproteinase
      NC1
      noncollagenous domain 1
      PI3-kinase
      phosphatidylinositol 3-kinase
      TGF-β1
      transforming growth factor TGF-β1
      IAP
      integrin-associated protein
      ALC
      anterior lens capsule
      PCR
      polymerase chain reaction
      RT-PCR
      reverse transcriptase-PCR
      IMDM
      Iscove's modified Dulbecco's medium
      FITC
      fluorescein isothiocyanate
      PBS
      phosphate-buffered saline
      PAGE
      polyacrylamide gel electrophoresis
      TBS
      Tris-buffered saline
      MTT
      3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
      Tumor cell invasion and metastasis are complex multistep processes that involve cell attachment to basement membrane or extracellular matrix proteins, degradation of adhesive proteins, and migration through the proteolytically modified tumor cell microenvironment (
      • Liotta L.A.
      • Stetler-Stevenson W.G.
      ,
      • Lester B.R.
      • McCarthy J.B.
      ). Anchorage of tumor cells to basement membrane proteins is mediated in part by integrins, a large family of heterodimeric cell surface receptors, that function not only as cell adhesion receptors but also as signaling receptors regulating cell growth, cell death, migration, and tissue remodeling (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ,
      • Clark E.A.
      • Brugge J.S.
      ). Since integrins are devoid of an intrinsic kinase activity, the outside-in signaling process initiated through ligand binding is thought to result in conformational changes of the receptor that are propagated from the ectodomain across the plasma membrane to the integrin cytoplasmic tails, allowing their interaction with intracellular signaling proteins. Integrin signaling leads to the activation of various kinases, such as focal adhesion kinase (FAK),1phosphatidylinositol 3-kinase (PI3-kinase), mitogen-activated protein kinase (
      • Schwartz M.A.
      • Schaller M.D.
      • Ginsberg M.H.
      ), as well as integrin-linked kinase (
      • Hannigan G.E.
      • Leung-Hagesteijn C.
      • Fitz-Gibbon L.
      • Coppolino M.G.
      • Radeva G.
      • Filmus J.
      • Bell J.C.
      • Dedhar S.
      ). Alternatively, integrin signaling can be mediated through transmembrane receptors that are associated with integrins in the cell membrane (
      • Hemler M.E.
      ). Integrin-associated protein (IAP) or CD47 is a widely expressed 50-kDa transmembrane protein that was initially identified through copurification with integrin αvβ3 from human placenta (
      • Brown E.
      • Hooper L.
      • Ho T.
      • Gresham H.
      ). CD47, which is composed of an N-terminal (extracellular) IgG variable domain, followed by five membrane-spanning hydrophobic helices and a cytoplasmic tail, is found in association with β3 and other integrins and has been implicated in multiple β3 integrin-mediated functions, such as enhanced αvβ3-dependent cell spreading or chemotaxis and, in platelets, αIIbβ3-dependent cell spreading and aggregation (
      • Frazier W.A.
      • Gao A.G.
      • Dimitry J.
      • Chung J.
      • Brown E.J.
      • Lindberg F.P.
      • Linder M.E.
      ). Both αvβ3 and IAP have been shown to stimulate FAK phosphorylation; however, in contrast to αvβ3, CD47-dependent downstream signaling does not involve PI3-kinase but appears to require a pertussis toxin-sensitive, heterotrimeric Gi protein, allowing activation of c-Src, which in turn phosphorylates SYK and FAK (
      • Frazier W.A.
      • Gao A.G.
      • Dimitry J.
      • Chung J.
      • Brown E.J.
      • Lindberg F.P.
      • Linder M.E.
      ,
      • Chung J.
      • Gao A.G.
      • Frazier W.A.
      ).
      A major component of basement membranes is type IV collagen, which forms their main structural framework and serves as scaffolding for the binding of other basement membrane proteins such as laminins, entactin, and proteoglycans (
      • Timpl R.
      • Brown J.C.
      ). Type IV collagen is an heterotrimer formed by three of six genetically distinct α chains (α1 to α6) (
      • Hudson B.G.
      • Reeders S.T.
      • Tryggvason K.
      ,
      • Mariyama M.
      • Leinonen A.
      • Mochizuki T.
      • Tryggvason K.
      • Reeders S.T.
      ,
      • Zhou J.
      • Ding M.
      • Zhao Z.
      • Reeders S.T.
      ). The α(IV) chains contain a typical collagenous domain of about 1400 amino acids and a COOH-terminal NC1 domain of about 230 amino acids. Type IV collagen and synthetic peptides derived from type IV collagen have been shown to promote cell adhesion, spreading, and migration (
      • Chelberg M.K.
      • McCarthy J.B.
      • Skubitz A.P.
      • Furcht L.T.
      • Tsilibary E.C.
      ,
      • Fields C.G.
      • Mickelson D.J.
      • Drake S.L.
      • McCarthy J.B.
      • Fields G.B.
      ,
      • Miles A.J.
      • Skubitz A.P.
      • Furcht L.T.
      • Fields G.B.
      ). We have previously shown that the anterior lens capsule (ALC) type IV collagen, which contains an α3(IV) chain, as well as the NC1 domain derived from the α3(IV) chain inhibited HT-144 melanoma cell proliferation and migration, whereas Engelbreth-Holm-Swarm collagen, which does not contain the α3(IV) chain, did not (
      • Pasco S.
      • Han J.
      • Gillery P.
      • Bellon G.
      • Maquart F.X.
      • Borel J.P.
      • Kefalides N.A.
      • Monboisse J.C.
      ,
      • Han J.
      • Ohno N.
      • Pasco S.
      • Monboisse J.C.
      • Borel J.P.
      • Kefalides N.A.
      ). Furthermore, within the NC1 domain of the α3(IV) chain, we have identified a peptide sequence, comprising residues 185–203, that is unique to the α3 chain and reproduces the same inhibitory effect on tumor cell proliferation as native ALC type IV collagen (
      • Han J.
      • Ohno N.
      • Pasco S.
      • Monboisse J.C.
      • Borel J.P.
      • Kefalides N.A.
      ). This peptide also inhibits tumor cell migration by down-regulating integrin αvβ3as well as the membrane type 1-matrix metalloproteinase (MT1-MMP), known to activate matrix metalloproteinase 2 (MMP-2) (
      • Pasco S.
      • Han J.
      • Gillery P.
      • Bellon G.
      • Maquart F.X.
      • Borel J.P.
      • Kefalides N.A.
      • Monboisse J.C.
      ). Although the precise physiological role of this α3(IV) collagen chain is still unknown, comparative analysis of the distribution of α1(IV) and α3(IV) chains in normal and neoplastic lung tissues revealed a selective localization of α3(IV) chains in alveolar basement membranes of normal lung tissue, in contrast to its pronounced localization at the interface between invasive tumor cell clusters and stroma in neoplastic lung tissue (
      • Polette M.
      • Thiblet J.
      • Ploton D.
      • Buisson A.C.
      • Monboisse J.C.
      • Tournier J.M.
      • Birembaut P.
      ), suggesting that one of the functional roles of α3(IV) chains could be to limit tumor development in the host tissue by inhibiting tumor cell proliferation.
      In an attempt to identify the tumor cell receptor interacting with the α3 chain of type IV collagen, we have previously identified the αvβ3-CD47 complex and three other proteins as the potential receptors for the α3(IV)179–208 peptide (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ). In the present study, we demonstrate that the α3(IV) peptide identifies a novel type IV collagen binding site on the β3 subunit of integrin αvβ3, distinct from the RGD recognition site, and initiates an αvβ3-dependent intracellular signaling process leading to FAK and PI3-kinase phosphorylation.

      EXPERIMENTAL PROCEDURES

      Reagents and Antibodies

      Horseradish peroxidase-conjugated sheep anti-mouse IgG was purchased from Amersham Pharmacia Biotech (Roosendaal, The Netherlands). Fluorescein-conjugated goat anti-mouse IgG and fluorescein-conjugated streptavidin were from Jackson Immunoresearch Laboratories Inc. (West Grove, PA). Anti-CD47 monoclonal antibody (mAb) (B6H12) was purchased from Pharmingen (San Diego, CA); polyclonal anti-FAK (C903) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); polyclonal anti-PI3-kinase p85 was from Upstate Biotechnologies, Inc. (Lake Placid, NY); monoclonal anti-FAK, anti-PI3-kinase p85, and anti-phosphotyrosine (PY-20) were from Transduction Laboratories (Lexington, KY); and anti-αv (VNR139) was from Life Technologies, Inc. (Merelbeke, Belgium). Human recombinant transforming growth factor β1 (TGF-β1) was purchased from R&D Systems (Minneapolis, MN). PCR primers were obtained from Eurogentec (Seraing, Belgium). The NC1 domain from ALC type IV collagen was prepared as described previously (
      • Monboisse J.C.
      • Garnotel R.
      • Bellon G.
      • Ohno N.
      • Perreau C.
      • Borel J.P.
      • Kefalides N.A.
      ). The biotinylated peptide corresponding to residues 185–206 in the NC1 domain of the α3 chain of type IV collagen,185CNYYSNSYSFWLASLNPERMFR206-KKK(Biotin)-NH2, as well as the corresponding scrambled peptide, MPSWRFASLEYCSRNFNYNYSL-KKK(Biotin)-NH2, were purchased from Neosystem (Strasbourg, France). The following monoclonal antibodies were generous gifts: anti-αIIb (S1.3) and anti-β3(4D10G3) (Dr. D. R. Phillips (COR Therapeutics, South San Francisco, CA)), anti-αvβ3 (23C6) (Dr. M. A. Horton (Bone and Mineral Center, Department of Medecine, University College London, United Kingdom)), and anti-LIBS1 and anti-LIBS2 (Dr. M. H. Ginsberg, Scripps Research Institute, La Jolla, CA).

      Cell Culture

      The human metastatic melanoma cell line HT-144 was a gift from Dr. P. Braquet (Bioinova, Plaisir, France). The Chinese hamster ovary (CHO) cell line CRL9096 was purchased from the American Type Culture Collection (ATCC, Manassas, VA). The CHO cell clones expressing human β3 (CHO A13), human αvβ3 (CHO A06), or human αIIbβ3 (CHO A10) have been established in our laboratory (
      • Kieffer N.
      • Melchior C.
      • Guinet J.M.
      • Michels S.
      • Gouon V.
      • Bron N.
      ). All cell lines were grown in Iscove's modified Dulbecco's medium (IMDM) (BioWhittaker, Verviers, Belgium), supplemented with 10% (v/v) heat-inactivated fetal calf serum, 2 mm glutamine, and penicillin-streptomycin (100 units/ml). Cells were routinely passaged with EDTA buffer, pH 7.4 (126 mm NaCl, 5 mm KCl, 1 mm EDTA, and 50 mm HEPES).

      Immunofluorescence and Flow Cytometry

      For flow cytometry analysis, cells were detached from culture plates with EDTA buffer and washed twice with IMDM. The cells (5 × 105) were then incubated for 30 min with the primary antibody or with the biotinylated α3(IV)185–206 peptide, washed with IMDM, and further incubated for 30 min with a FITC-conjugated goat anti-mouse secondary antibody or with FITC-conjugated streptavidin. Cells were then washed and resuspended in phosphate-buffered saline (PBS) (136 mm NaCl, 2.7 mm KOH, 8 mm Na2HPO4, 1.8 mmKH2PO4, pH 7.4) and subsequently analyzed on an Epics Elite ESP flow cytometer (Coulter Corp., Hialeah, FL). For immunofluorescence microscopy, the labeled cells were fixed by methanol on a glass coverslip, mounted in PBS-glycerol, and examined with a Leica DMRB microscope using a × 63 oil immersion objective.

      Immunoprecipitation and Western Blot Analysis

      Preparation of Cell Lysates

      Cells were detached with EDTA buffer, washed twice in cold PBS, and lysed for 30 min in ice-cold lysis buffer A (10 mm Tris-HCl, pH 7.4, 150 mmNaCl, 1% Triton X-100, 5 mm phenylmethylsulfonyl fluoride). Lysates were cleared by centrifugation at 12,000 ×g for 10 min at 4 °C, and the protein concentration was determined using Lowry's modified method (
      • Dulley J.R.
      • Grieve P.A.
      ). For the identification of phosphotyrosine proteins in total cell extracts, HT-144 melanoma cells were resuspended in IMDM and serum-starved for 90 min at 37 °C. The cells (106/ml) were then incubated with the α3(IV)185–206 peptide (20 μg/ml) or the corresponding scrambled peptide for different time points, centrifuged for 2 min at 1200 × g, and lysed with 2% SDS in lysis buffer B (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 5 mm EDTA, 10 mm sodium fluoride, 2 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin and aprotinin). Lysates were immediately heated at 90 °C for 5 min prior to protein concentration determination.

      Immunoprecipitation

      Cell lysate (1 mg of protein) was incubated overnight at 4 °C with the anti-αvβ3 mAb 23C6 or the anti-CD47 mAb B6H12. Immune complexes were precipitated with protein A-Sepharose beads for 1 h at 4 °C. The beads were then washed three times with lysis buffer A, and the precipitates were recovered by boiling the beads in 30 μl of SDS sample buffer (125 mm Tris-HCl, pH 6.8, 4.6% SDS, 20% glycerol, 0.5 mg/ml bromphenol blue). For phosphotyrosine protein immunoprecipitation, HT-144 melanoma cells were incubated with the α3(IV)185–206 peptide as described above and lysed with 1% Triton X-100 and 0.1% sodium deoxycholate in lysis buffer B. Cell extracts (1 mg of protein) were incubated overnight at 4 °C with either the polyclonal anti-FAK or the anti-PI3-kinase antibody, and the immune complexes were processed as described above.

      Western Blot Analysis

      Immunoprecipitates or total cell lysates (50 μg of protein) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose Hybond C membrane (Amersham Pharmacia Biotech) using a semidry transblot apparatus (Amersham Pharmacia Biotech). The membranes were blocked overnight in Tris-buffered saline (TBS) (20 mm Tris-HCl, pH 7.4, 137 mm NaCl) containing 0.1% Tween and 5% nonfat dry milk and subsequently incubated for 2 h at room temperature with the anti-β3 mAb 4D10G3 or the anti-CD47 mAb B6H12. Following several washes in TBS-Tween, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat anti-mouse IgG in TBS-Tween containing 5% nonfat dry milk, washed in TBS-Tween, and finally processed using a chemiluminescence kit (SuperSignal; Pierce). For membrane stripping, the membranes were incubated at 50 °C in stripping buffer (62.5 mm Tris, pH 6.7, 2% SDS, and 100 mm β-mercaptoethanol) and extensively washed. For identification of phosphotyrosine proteins, the blots were blocked with TBS containing 0.1% Tween and 1% bovine serum albumin and probed with the anti-phosphotyrosine mAb PY-20.

      Liquid Phase Receptor Capture Assay Using the Biotinylated α3(IV)185–206 Peptide

      Cell extracts (2 mg of protein) were incubated overnight at 4 °C with 5 μg of the biotinylated α3(IV)185–206 peptide in 10 mm Tris-HCl, 150 mm NaCl, 1 mmCaCl2, 1 mm MgCl2, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and aprotinin. Streptavidin-Sepharose beads were then added and incubated for 1 h at 4 °C. The beads were extensively washed with the same buffer, and the captured receptors were recovered by boiling the beads in 30 μl of SDS sample buffer. Recovered proteins were resolved by SDS-PAGE and transferred onto nitrocellulose. The membranes were blocked and then incubated with either the anti-β3 (4D10G3), anti-αv (VNR139), or anti-αIIb (S1.3) mAbs as described above.

      Adhesion Assay

      96-Well plates were coated overnight at 4 °C with fibrinogen (20 μg/ml in PBS buffer), the NC1 domain of ALC type IV collagen (20 μg/ml), or the α3(IV)185–206 peptide (20 μg/ml in 0.05m sodium carbonate/sodium bicarbonate buffer, pH 9.6) and then blocked for 1 h at 37 °C with 20 mg/ml of sterile-filtered, heat-inactivated bovine serum albumin in PBS. Prior to use, the plates were washed twice with PBS. The cells were detached with EDTA buffer, washed, preincubated for 15 min with RGDS (2 mm) or the α3(IV)185–206 peptide (20 μm), and subsequently transferred to the wells (30,000 cells/well). After a 2-h incubation at 37 °C, individual microtiter wells were microphotographed using a Nikon inverted microscope equipped with phase contrast.

      Cell Proliferation Assay

      Cells were detached with EDTA buffer, washed and resuspended in IMDM, and then added to 96-well plates coated with poly-l-lysine. After a 3-h incubation at 37 °C, the peptide (20 μg/ml in IMDM) was added to the wells, and the cells were further incubated for 48 h at 37 °C. At the end of the incubation period, the cells were quantitated using the modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (
      • Loveland B.E.
      • Johns T.G.
      • Mackay I.R.
      • Vaillant F.
      • Wang Z.X.
      • Hertzog P.J.
      ). Briefly, MTT was added to each well to a final concentration of 0.5 mg/ml. After a 4-h incubation at 37 °C, the culture medium was removed, and 100 μl of Me2SO was added to each well. The absorbance of the samples was measured at 570 nm. A MTT calibration curve was performed with samples of known cell concentration to correlate the measured absorbance with cell number. Each assay was carried out in triplicate.

      Semiquantitative RT-PCR

      Total RNA was extracted with guanidinium/phenol/chloroform (
      • Chomczynski P.
      • Sacchi N.
      ). For semiquantitative RT-PCR, 2 μg of total RNA were transcribed to cDNA with an RNA PCR kit (TaKaRa Biomedicals) using an antisense primer for MT1-MMP together with the antisense primer β-actin used here as an internal control. The MT1-MMP cDNA was then amplified together with the β-actin cDNA in a single tube for 25 cycles. The following antisense primers were used: 5′-ACATCAAAGTCTGGGAAGGA-3′ for MT1-MMP and 5′-GACGATGCCGTGCTCGATG-3′ for β-actin. Sense primers were 5′-AGCAGGGAACGCTGGCAGT-3′ for MT1-MMP and 5′-GATCCGCCGCCCGTCCACA-3′ for β-actin. cDNA amplification was performed on a Perkin-Elmer GeneAmp PCR System 2400, and the cycling program included a 20-s denaturation step at 95 °C and a 30-s annealing step at 55 °C, followed by a 30-s elongation step at 72 °C. The RT-PCR cDNA products were electrophoresed through a 1.5% agarose gel and visualized after ethidium bromide staining of the gel.

      DISCUSSION

      Basement membranes are thin specialized extracellular matrices that are functionally important for embryonic development, maintenance of tissue architecture, tissue remodeling during development and wound healing, and protection of tissues and organs from mechanical stress or exogenous factors (
      • Sado Y.
      • Kagawa M.
      • Naito I.
      • Ueki Y.
      • Seki T.
      • Momota R.
      • Oohashi T.
      • Ninomiya Y.
      ). A major component of all basement membranes is type IV collagen (
      • Timpl R.
      • Brown J.C.
      ), which promotes cell adhesion and migration (
      • Chelberg M.K.
      • McCarthy J.B.
      • Skubitz A.P.
      • Furcht L.T.
      • Tsilibary E.C.
      ,
      • Fields C.G.
      • Mickelson D.J.
      • Drake S.L.
      • McCarthy J.B.
      • Fields G.B.
      ,
      • Miles A.J.
      • Skubitz A.P.
      • Furcht L.T.
      • Fields G.B.
      ). Tumor cell interactions with type IV collagen have been shown to rely on α3β1 or αvβ3 integrins, leading to changes in their invasive properties as well as changes in their expression of various MMPs, such as MMP-1 or MMP-2 (
      • Lauer J.L.
      • Gendron C.M.
      • Fields G.B.
      ,
      • Seftor R.E.
      • Seftor E.A.
      • Gehlsen K.R.
      • Stetler-Stevenson W.G.
      • Brown P.D.
      • Ruoslahti E.
      • Hendrix M.J.
      ). We have previously reported that a peptide sequence corresponding to residues 185–203 in the α3(IV) chain of type IV collagen was able to inhibit in vitro tumor cell proliferation and migration through reconstituted basement membranes. The peptide sequence contains a SNS triplet in position 189–191 that is unique to the α3(IV) collagen chain, and replacement of each of the two serine residues by an alanine abolished its biological activity (
      • Pasco S.
      • Han J.
      • Gillery P.
      • Bellon G.
      • Maquart F.X.
      • Borel J.P.
      • Kefalides N.A.
      • Monboisse J.C.
      ,
      • Han J.
      • Ohno N.
      • Pasco S.
      • Monboisse J.C.
      • Borel J.P.
      • Kefalides N.A.
      ). The cysteine residue at position 185 is also required for the biological activity of the α3(IV) peptide. Engelbreth-Holm-Swarm type IV collagen, which lacks the α3(IV) chain, failed to inhibit tumor cell proliferation and migration. Finally, a tentative affinity chromatography of melanoma cell extract on a α3(IV)179–208 peptide column identified the αvβ3-CD47 complex as the tumor cell receptors (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ).
      In the present study, we have characterized in detail the α3(IV)185–206 peptide binding site on the αvβ3-CD47 complex and provide evidence that the peptide specifically interacts with the β subunit of integrin αvβ3. Indeed, cells expressing αvβ3 interacted with the α3(IV)185–206 peptide, independently of the presence or absence of CD47, whereas cells expressing CD47 in the absence of αvβ3 did not. Furthermore, to demonstrate a direct interaction of αvβ3 with the α3(IV)185–206 peptide, we performed receptor capture experiments using the biotinylated α3(IV)185–206 peptide and cell extracts of various cell types. When HT-144 melanoma cell extract was used, the αvβ3-CD47 complex was recovered, in accordance with our previous data (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ). The α3(IV)185–206 peptide also interacted with αvβ3 in the absence of CD47, whereas it was unable to capture CD47 in the absence of αvβ3. Since we have previously shown that the blocking anti-CD47 mAb B6H12 partially abolished the inhibitory effect of the α3(IV) peptide on tumor cell proliferation (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ), a possible explanation for this result could be an antibody-dependent steric hindrance of the αvβ3/peptide interaction. Indeed, data by Gresham et al. (
      • Gresham H.D.
      • Goodwin J.L.
      • Allen P.M.
      • Anderson D.C.
      • Brown E.J.
      ) have provided evidence that the anti-CD47 mAb B6H12 specifically inhibits the enhancement of neutrophil phagocytosis by inhibiting the RGD-dependent integrin/ligand interaction, although affinity-purified CD47 was unable to interact with the RGD sequence. Alternatively, the blocking anti-CD47 mAb could induce conformational changes of αvβ3, preventing its interaction with the α3(IV) peptide. This hypothesis is supported by data showing that stimulation of CD47 by its agonist 4N1K, a peptide derived from the COOH-terminal cell binding domain of thrombospondin, spontaneously activates αIIbβ3, as demonstrated by enhanced binding of the conformationally sensitive mAb PAC-1 (
      • Chung J.
      • Gao A.G.
      • Frazier W.A.
      ).
      Integrin αvβ3 constitutively interacts with a large variety of RGD-containing adhesive proteins present in the extracellular matrix (
      • Cheresh D.A.
      • Spiro R.C.
      ), and this interaction is mediated through the metal ion-dependent adhesion site-like domain of the β3 subunit (
      • Tozer E.C.
      • Liddington R.C.
      • Sutcliffe M.J.
      • Smeeton A.H.
      • Loftus J.C.
      ). By using different cell lines expressing either αvβ3 or αIIbβ3, we provide evidence that the α3(IV) peptide binds to the β3 subunit. However, the α3(IV) peptide binding site is clearly distinct from the RGD recognition site, since inhibition experiments with an RGDS peptide did not inhibit melanoma cell adhesion to immobilized α3(IV)185–206 peptide, whereas RGDS completely inhibited melanoma and CHO αvβ3 cell adhesion to immobilized fibrinogen.
      Ligand binding to β3 integrins is known to induce conformational changes of the integrin receptor ectodomain, reflecting the most earliest events in integrin-dependent outside-in signaling. These conformational changes can be monitored with monoclonal antibodies to neoepitopes known as LIBS (
      • Pelletier A.J.
      • Kunicki T.
      • Quaranta V.
      ). Interestingly, similar to RGDS, the α3(IV)185–206 peptide was able to induce LIBS epitope expression on the β3 integrin subunit. More surprisingly, however, the α3(IV) peptide, in contrast to the RGDS peptide, was also able to trigger intracellular signaling processes in melanoma cells in suspension, such as tyrosine phosphorylation of FAK and PI3-kinase. There is convincing evidence that a dimeric ligand is necessary to initiate β3 integrin-dependent intracellular tyrosine phosphorylation, since the monomeric cell recognition peptide RGDS fails to cause intracellular tyrosine phosphorylation (
      • Miyamoto S.
      • Akiyama S.K.
      • Yamada K.M.
      ,
      • Miyamoto S.
      • Teramoto H.
      • Coso O.A.
      • Gutkind J.S.
      • Burbelo P.D.
      • Akiyama S.K.
      • Yamada K.M.
      ), despite the fact that RGDS binds to the receptor and induces conformational changes of its ectodomain (
      • Frelinger III, A.L.
      • Cohen I.
      • Plow E.F.
      • Smith M.A.
      • Roberts J.
      • Lam S.C.
      • Ginsberg M.H.
      ,
      • Juliano D.
      • Wang Y.
      • Marcinkiewicz C.
      • Rosenthal L.A.
      • Stewart G.J.
      • Niewiarowski S.
      ). Bhattacharya et al. (
      • Bhattacharya S.
      • Fu C.
      • Bhattacharya J.
      • Greenberg S.
      ) have shown that monomeric vitronectin does not induce enhanced protein tyrosine phosphorylation in bovine pulmonary artery endothelial cells, whereas multimeric vitronectin elicites time- and concentration-dependent increases in tyrosine phosphorylation of proteins such as FAK, paxillin, Shc, cortactin, or ezrin. Since the α3(IV) peptide contains a cysteine at position 185 that is essential for its biological activity, one possible explanation is that the α3(IV) peptide, through its binding to αvβ3, allows the establishment of disulfide bonds that induce clustering of the αvβ3 integrin, necessary for outside-in signaling. Such receptor clustering can indeed be observed on the microphotographs of HT-144 and CHO αvβ3cells incubated with the biotinylated α3(IV) peptide.
      In a previous report, we have provided evidence that inhibition of tumor cell proliferation by the α3(IV) peptide relies on elevated levels of cAMP and involves cAMP-dependent protein kinase A (
      • Shahan T.A.
      • Ohno N.
      • Pasco S.
      • Borel J.P.
      • Monboisse J.C.
      • Kefalides N.A.
      ). Since PI3-kinase has previously been shown to activate protein kinase A in a cAMP-dependent manner (
      • Edinger R.S.
      • Rokaw M.D.
      • Johnson J.P.
      ), we wondered whether PI3-kinase could be involved in the signaling pathway, leading to inhibition of melanoma cell proliferation. The fact that pretreatment of HT-144 and CHO αvβ3 cells with wortmannin, a PI3-kinase inhibitor, completely reverted the inhibitory effect of the α3(IV) peptide on cell proliferation, confirms this hypothesis. Our previously published data have also shown that the α3(IV) peptide inhibits tumor cell migration by decreasing MT1-MMP expression. MT1-MMP is known to activate latent MMP-2 (
      • Lichte A.
      • Kolkenbrock H.
      • Tschesche H.
      ), thereby facilitating matrix degradation and cellular invasion. Also, since data by Yu et al. (
      • Yu M.
      • Sato H.
      • Seiki M.
      • Spiegel S.
      • Thompson E.W.
      ) have shown that an increase in intracellular cAMP inhibits MT1-MMP expression, we studied the possible involvement of PI3-kinase in this signaling pathway. Pretreatment of HT-144 melanoma cells with wortmannin suppressed the inhibitory effect induced by the α3(IV) peptide on MT1-MMP gene expression, as demonstrated by semiquantitative RT-PCR analysis, providing evidence that PI3-kinase is involved in the α3(IV) peptide signaling pathway leading to an inhibition of MT1-MMP gene expression. These results are in good agreement with those of Esparza et al. (
      • Esparza J.
      • Vilardell C.
      • Calvo J.
      • Juan M.
      • Vives J.
      • Urbano-Marquez A.
      • Yague J.
      • Cid M.C.
      ), who reported that inhibition of PI3-kinase by wortmannin strongly increased fibronectin-induced MMP production. Taken together, our results highlight a new αvβ3-dependent signaling pathway initiated through collagen type IV binding to the integrin β3 subunit that stimulates FAK and PI3-kinase activation. PI3-kinase, by activating adenylate cyclase, could thus represent the missing link in the signaling pathway leading to cAMP-dependent inhibition of tumor cell proliferation.

      Acknowledgments

      We thank Drs D. Phillips, M. Horton, and M. H. Ginsberg for generous gifts of monoclonal antibodies. We also thank W. Ammerlaan and N. H. C. Brons for expert assistance with flow cytometry analysis.

      REFERENCES

        • Liotta L.A.
        • Stetler-Stevenson W.G.
        Cancer Res. 1991; 51: 5054s-5059s
        • Lester B.R.
        • McCarthy J.B.
        Cancer Metastasis Rev. 1992; 11: 31-44
        • Schwartz M.A.
        • Schaller M.D.
        • Ginsberg M.H.
        Annu. Rev. Cell Dev. Biol. 1995; 11: 549-599
        • Clark E.A.
        • Brugge J.S.
        Science. 1995; 268: 233-239
        • Hannigan G.E.
        • Leung-Hagesteijn C.
        • Fitz-Gibbon L.
        • Coppolino M.G.
        • Radeva G.
        • Filmus J.
        • Bell J.C.
        • Dedhar S.
        Nature. 1996; 379: 91-96
        • Hemler M.E.
        Curr. Opin. Cell Biol. 1998; 10: 578-585
        • Brown E.
        • Hooper L.
        • Ho T.
        • Gresham H.
        J. Cell Biol. 1990; 111: 2785-2794
        • Frazier W.A.
        • Gao A.G.
        • Dimitry J.
        • Chung J.
        • Brown E.J.
        • Lindberg F.P.
        • Linder M.E.
        J. Biol. Chem. 1999; 274: 8554-8560
        • Chung J.
        • Gao A.G.
        • Frazier W.A.
        J. Biol. Chem. 1997; 272: 14740-14746
        • Timpl R.
        • Brown J.C.
        Bioessays. 1996; 18: 123-132
        • Hudson B.G.
        • Reeders S.T.
        • Tryggvason K.
        J. Biol. Chem. 1993; 268: 26033-26036
        • Mariyama M.
        • Leinonen A.
        • Mochizuki T.
        • Tryggvason K.
        • Reeders S.T.
        J. Biol. Chem. 1994; 269: 23013-23017
        • Zhou J.
        • Ding M.
        • Zhao Z.
        • Reeders S.T.
        J. Biol. Chem. 1994; 269: 13193-13199
        • Chelberg M.K.
        • McCarthy J.B.
        • Skubitz A.P.
        • Furcht L.T.
        • Tsilibary E.C.
        J. Cell Biol. 1990; 111: 261-270
        • Fields C.G.
        • Mickelson D.J.
        • Drake S.L.
        • McCarthy J.B.
        • Fields G.B.
        J. Biol. Chem. 1993; 268: 14153-14160
        • Miles A.J.
        • Skubitz A.P.
        • Furcht L.T.
        • Fields G.B.
        J. Biol. Chem. 1994; 269: 30939-30945
        • Pasco S.
        • Han J.
        • Gillery P.
        • Bellon G.
        • Maquart F.X.
        • Borel J.P.
        • Kefalides N.A.
        • Monboisse J.C.
        Cancer Res. 2000; 60: 467-473
        • Han J.
        • Ohno N.
        • Pasco S.
        • Monboisse J.C.
        • Borel J.P.
        • Kefalides N.A.
        J. Biol. Chem. 1997; 272: 20395-20401
        • Polette M.
        • Thiblet J.
        • Ploton D.
        • Buisson A.C.
        • Monboisse J.C.
        • Tournier J.M.
        • Birembaut P.
        J. Pathol. 1997; 182: 185-191
        • Shahan T.A.
        • Ziaie Z.
        • Pasco S.
        • Fawzi A.
        • Bellon G.
        • Monboisse J.C.
        • Kefalides N.A.
        Cancer Res. 1999; 59: 4584-4590
        • Monboisse J.C.
        • Garnotel R.
        • Bellon G.
        • Ohno N.
        • Perreau C.
        • Borel J.P.
        • Kefalides N.A.
        J. Biol. Chem. 1994; 269: 25475-25482
        • Kieffer N.
        • Melchior C.
        • Guinet J.M.
        • Michels S.
        • Gouon V.
        • Bron N.
        Cell Adhes. Commun. 1996; 4: 25-39
        • Dulley J.R.
        • Grieve P.A.
        Anal. Biochem. 1975; 64: 136-141
        • Loveland B.E.
        • Johns T.G.
        • Mackay I.R.
        • Vaillant F.
        • Wang Z.X.
        • Hertzog P.J.
        Biochem. Int. 1992; 27: 501-510
        • Chomczynski P.
        • Sacchi N.
        Anal. Biochem. 1987; 162: 156-159
        • Janji B.
        • Melchior C.
        • Gouon V.
        • Vallar L.
        • Kieffer N.
        Int. J. Cancer. 1999; 83: 255-262
        • Frelinger III, A.L.
        • Cohen I.
        • Plow E.F.
        • Smith M.A.
        • Roberts J.
        • Lam S.C.
        • Ginsberg M.H.
        J. Biol. Chem. 1990; 265: 6346-6352
        • Pampori N.
        • Hato T.
        • Stupack D.G.
        • Aidoudi S.
        • Cheresh D.A.
        • Nemerow G.R.
        • Shattil S.J.
        J. Biol. Chem. 1999; 274: 21609-21616
        • Sado Y.
        • Kagawa M.
        • Naito I.
        • Ueki Y.
        • Seki T.
        • Momota R.
        • Oohashi T.
        • Ninomiya Y.
        J. Biochem. (Tokyo ). 1998; 123: 767-776
        • Lauer J.L.
        • Gendron C.M.
        • Fields G.B.
        Biochemistry. 1998; 37: 5279-5287
        • Seftor R.E.
        • Seftor E.A.
        • Gehlsen K.R.
        • Stetler-Stevenson W.G.
        • Brown P.D.
        • Ruoslahti E.
        • Hendrix M.J.
        Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1557-1561
        • Gresham H.D.
        • Goodwin J.L.
        • Allen P.M.
        • Anderson D.C.
        • Brown E.J.
        J. Cell Biol. 1989; 108: 1935-1943
        • Cheresh D.A.
        • Spiro R.C.
        J. Biol. Chem. 1987; 262: 17703-17711
        • Tozer E.C.
        • Liddington R.C.
        • Sutcliffe M.J.
        • Smeeton A.H.
        • Loftus J.C.
        J. Biol. Chem. 1996; 271: 21978-21984
        • Pelletier A.J.
        • Kunicki T.
        • Quaranta V.
        J. Biol. Chem. 1996; 271: 1364-1370
        • Miyamoto S.
        • Akiyama S.K.
        • Yamada K.M.
        Science. 1995; 267: 883-885
        • Miyamoto S.
        • Teramoto H.
        • Coso O.A.
        • Gutkind J.S.
        • Burbelo P.D.
        • Akiyama S.K.
        • Yamada K.M.
        J. Cell Biol. 1995; 131: 791-805
        • Juliano D.
        • Wang Y.
        • Marcinkiewicz C.
        • Rosenthal L.A.
        • Stewart G.J.
        • Niewiarowski S.
        Exp. Cell Res. 1996; 225: 132-142
        • Bhattacharya S.
        • Fu C.
        • Bhattacharya J.
        • Greenberg S.
        J. Biol. Chem. 1995; 270: 16781-16787
        • Shahan T.A.
        • Ohno N.
        • Pasco S.
        • Borel J.P.
        • Monboisse J.C.
        • Kefalides N.A.
        Connect. Tissue Res. 1999; 40: 221-232
        • Edinger R.S.
        • Rokaw M.D.
        • Johnson J.P.
        Am. J. Physiol. 1999; 277: F575-F579
        • Lichte A.
        • Kolkenbrock H.
        • Tschesche H.
        FEBS Lett. 1996; 397: 277-282
        • Yu M.
        • Sato H.
        • Seiki M.
        • Spiegel S.
        • Thompson E.W.
        Clin. Exp. Metastasis. 1998; 16: 185-191
        • Esparza J.
        • Vilardell C.
        • Calvo J.
        • Juan M.
        • Vives J.
        • Urbano-Marquez A.
        • Yague J.
        • Cid M.C.
        Blood. 1999; 94: 2754-2766