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Laminin Receptor Involvement in the Anti-angiogenic Activity of Pigment Epithelium-derived Factor

2009; Elsevier BV; Volume: 284; Issue: 16 Linguagem: Inglês

10.1074/jbc.m809259200

ISSN

1083-351X

Autores

Adrien Bernard, Jacqueline Gao-Li, Cláudio A. Franco, Tahar Bouceba, Alexis Huet, Zhenlin Li,

Tópico(s)

melanin and skin pigmentation

Resumo

Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A2/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44–77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120–210). A 25-mer peptide named P46 (aa 46–70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition. Pigment epithelium-derived factor (PEDF) is a multifunctional protein with neurotrophic, anti-oxidative, and anti-inflammatory properties. It is also one of the most potent endogenous inhibitors of angiogenesis, playing an important role in restricting tumor growth, invasion, and metastasis. Studies show that PEDF binds to cell surface proteins, but little is known about how it exerts its effects. Recently, research identified phospholipase A2/nutrin/patatin-like phospholipase domain-containing 2 as one PEDF receptor. To identify other receptors, we performed yeast two-hybrid screening using PEDF as bait and discovered that the non-integrin 37/67-kDa laminin receptor (LR) is another PEDF receptor. Co-immunoprecipitation, His tag pulldown, and surface plasmon resonance assays confirmed the interaction between PEDF and LR. Using the yeast two-hybrid method, we further restricted the LR-interacting domain on PEDF to a 34-amino acid (aa) peptide (aa 44–77) and the PEDF-interacting domain on LR to a 91-aa fragment (aa 120–210). A 25-mer peptide named P46 (aa 46–70), derived from 34-mer, interacts with LR in surface plasmon resonance assays and binds to endothelial cell (EC) membranes. This peptide induces EC apoptosis and inhibits EC migration, tube-like network formation in vitro, and retinal angiogenesis ex vivo, like PEDF. Our results suggest that LR is a real PEDF receptor that mediates PEDF angiogenesis inhibition. Pigmented epithelium-derived factor (PEDF), 2The abbreviations used are: PEDF, pigment epithelium-derived factor; AD, activation domain; BD, binding domain; KAP, keratin-associated protein; LR laminin receptor; PNPLA2, PLA2/nutrin/patatin-like phospholipase domain-containing 2; RU, resonance unit; TUNEL, terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling; Y2H, yeast two-hybrid; aa, amino acid(s); HuBMEC, human bone marrow endothelial cell; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; MAPK, mitogen-activated protein kinase; JNK, c-Jun N-terminal kinase; FBS, fetal bovine serum; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; DAPI, 4′,6′-diamino-2-phenylindole; SPR, surface plasmon resonance; siRNA, small interfering RNA; HA, hemagglutinin; EC, endothelial cell; X-α-Gal, 5′-bromo-4-chloro-3′-indolyl-α-d-galactopyranoside. also known as SERPIN F1 and EPC1, is a 50-kDa serpin-like peptide. Although first identified in cultured pigment epithelial cells from fetal human retinas (1Tombran-Tink J. Chader G.G. Johnson L.V. Exp. Eye Res... 1991; 53: 411-414Google Scholar), we now know that liver, kidney, heart, testis, and lung tissues also express PEDF (2Tombran-Tink J. Mazuruk K. Rodriguez I.R. Chung D. Linker T. Englander E. Chader G.J. Mol. Vis... 1996; 2: 11Google Scholar). PEDF influences many biological processes. It is anti-angiogenic, anti-tumorigenic, anti-inflammatory, anti-oxidative, neurotrophic, and neuroprotective, and it exhibits anti-vasopermeability properties (3Bouck N. Trends Mol. Med... 2002; 8: 330-334Google Scholar, 4Tombran-Tink J. Barnstable C.J. Nat. Rev. Neurosci... 2003; 4: 628-636Google Scholar, 5Becerra S.P. Exp. Eye Res... 2006; 82: 739-740Google Scholar, 6Ek E.T. Dass C.R. Choong P.F. Trends Mol. Med... 2006; 12: 497-502Google Scholar, 7Zhang S.X. Wang J.J. Gao G. Shao C. Mott R. Ma J.X. FASEB J... 2006; 20: 323-325Google Scholar, 8Yamagishi S. Nakamura K. Ueda S. Kato S. Imaizumi T. Cell Tissue Res... 2005; 320: 437-445Google Scholar-9Liu H. Ren J.G. Cooper W.L. Hawkins C.E. Cowan M.R. Tong P.Y. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 6605-6610Google Scholar). These diverse actions affect many cell types, including retinal cells (10Yamagishi S. Inagaki Y. Amano S. Okamoto T. Takeuchi M. Makita Z. Biochem. Biophys. Res. Commun... 2002; 296: 877-882Google Scholar), neuronal cells (11Steele F.R. Chader G.J. Johnson L.V. Tombran-Tink J. Proc. Natl. Acad. Sci. U. S. A... 1993; 90: 1526-1530Google Scholar), endothelial cells (12Stellmach V. Crawford S.E. Zhou W. Bouck N. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 2593-2597Google Scholar), and hematopoietic stem cells (13Selleri C. Ragno P. Ricci P. Visconte V. Scarpato N. Carriero M.V. Rotoli B. Rossi G. Montuori N. Blood.. 2006; 108: 2476-2484Google Scholar). X-ray diffraction studies show that PEDF has an asymmetrical charge distribution (14Simonovic M. Gettins P.G. Volz K. Proc. Natl. Acad. Sci. U. S. A... 2001; 98: 11131-11135Google Scholar). A high density of basic residues on one side of the molecule (positive) interact with heparin and glycosaminoglycans, whereas acidic residues on the opposite side (negative) interact with type-1 collagen (15Alberdi E. Hyde C.C. Becerra S.P. Biochemistry.. 1998; 37: 10643-10652Google Scholar, 16Yasui N. Mori T. Morito D. Matsushita O. Kourai H. Nagata K. Koide T. Biochemistry.. 2003; 42: 3160-3167Google Scholar, 17Becerra S.P. Perez-Mediavilla L.A. Weldon J.E. Locatelli-Hoops S. deS Senanayake P. Notari L. Notario V. Hollyfield J.G. J. Biol. Chem... 2008; 283: 33310-33320Google Scholar, 18Meyer C. Notari L. Becerra S.P. J. Biol. Chem... 2002; 277: 45400-45407Google Scholar-19Hosomichi J. Yasui N. Koide T. Soma K. Morita I. Biochem. Biophys. Res. Commun... 2005; 335: 756-761Google Scholar). Yet the mechanisms explaining the diverse biological activities of PEDF remain unclear. A ligand/receptor interaction at the cell membrane seemed likely, in addition to interactions within extracellular matrices, because of the diverse effects and ubiquitous expression of PEDF and the fact that most PEDF deposits remain within extracellular matrices (20Alberdi E.M. Weldon J.E. Becerra S.P. BMC Biochem... 2003; 4: 1Google Scholar). We suspected that distinct PEDF receptors elicit divergent signals to cause different biological effects. Indeed, evidence shows that PEDF binds at least two receptors: a 60-kDa receptor in ECs and an 80-kDa receptor in neuronal cells (21Alberdi E. Aymerich M.S. Becerra S.P. J. Biol. Chem... 1999; 274: 31605-31612Google Scholar, 22Bilak M.M. Becerra S.P. Vincent A.M. Moss B.H. Aymerich M.S. Kuncl R.W. J. Neurosci... 2002; 22: 9378-9386Google Scholar, 23Aymerich M.S. Alberdi E.M. Martínez A. Becerra S.P. Investig. Ophthalmol. Vis. Sci... 2001; 42: 3287-3293Google Scholar-24Yamagishi S. Inagaki Y. Nakamura K. Abe R. Shimizu T. Yoshimura A. Imaizumi T. J. Mol. Cell Cardiol... 2004; 37: 497-506Google Scholar). Research has identified two functional epitopes on PEDF: a 34-mer peptide (residues 44–77) and a 44-mer peptide (residues 78–121) (25Filleur S. Volz K. Nelius T. Mirochnik Y. Huang H. Zaichuk T.A. Aymerich M.S. Becerra S.P. Yap R. Veliceasa D. Shroff E.H. Volpert O.V. Cancer Res... 2005; 65: 5144-5152Google Scholar). The 44-mer peptide interacts with the putative 80-kDa receptor identified on Y-79 cells and cerebellar, motor (21Alberdi E. Aymerich M.S. Becerra S.P. J. Biol. Chem... 1999; 274: 31605-31612Google Scholar, 22Bilak M.M. Becerra S.P. Vincent A.M. Moss B.H. Aymerich M.S. Kuncl R.W. J. Neurosci... 2002; 22: 9378-9386Google Scholar), and retinal (23Aymerich M.S. Alberdi E.M. Martínez A. Becerra S.P. Investig. Ophthalmol. Vis. Sci... 2001; 42: 3287-3293Google Scholar) neurons, to replicate the neurotrophic and anti-vasopermeability properties of PEDF (9Liu H. Ren J.G. Cooper W.L. Hawkins C.E. Cowan M.R. Tong P.Y. Proc. Natl. Acad. Sci. U. S. A... 2004; 101: 6605-6610Google Scholar, 25Filleur S. Volz K. Nelius T. Mirochnik Y. Huang H. Zaichuk T.A. Aymerich M.S. Becerra S.P. Yap R. Veliceasa D. Shroff E.H. Volpert O.V. Cancer Res... 2005; 65: 5144-5152Google Scholar). Becerra and co-workers (26Notari L. Baladron V. Aroca-Aguilar J.D. Balko N. Heredia R. Meyer C. Notario P.M. Saravanamuthu S. Nueda M.L. Sanchez-Sanchez F. Escribano J. Laborda J. Becerra S.P. J. Biol. Chem... 2006; 281: 38022-38037Google Scholar) recently identified 80-kDa PLA2/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) as a PEDF receptor that binds the 44-mer epitope. Filleur et al. (25Filleur S. Volz K. Nelius T. Mirochnik Y. Huang H. Zaichuk T.A. Aymerich M.S. Becerra S.P. Yap R. Veliceasa D. Shroff E.H. Volpert O.V. Cancer Res... 2005; 65: 5144-5152Google Scholar) showed in vivo that overexpressing the 34-mer in PC-3 prostate adenocarcinoma cell lines reduces tumor microvessel density and increases apoptosis. The 34-mer peptide induces apoptosis, blocks endothelial cell migration, and inhibits corneal angiogenesis, possibly through a distinct EC receptor. We do not yet know the nature of the 60-kDa receptor. PEDF is one of the most potent natural endogenous inhibitors of angiogenesis, the extension of the vascular network from pre-existing blood vessels. It inhibits endothelial cell migration even in the presence of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) (27Duh E.J. Yang H.S. Suzuma I. Miyagi M. Youngman E. Mori K. Katai M. Yan L. Suzuma K. West K. Davarya S. Tong P. Gehlbach P. Pearlman J. Crabb J.W. Aiello L.P. Campochiaro P.A. Zack D.J. Investig. Ophthalmol. Vis. Sci... 2002; 43: 821-829Google Scholar, 28Hutchings H. Maitre-Boube M. Tombran-Tink J. Plouët J. Biochem. Biophys. Res. Commun... 2002; 294: 764-769Google Scholar-29Kanda S. Mochizuki Y. Nakamura T. Miyata Y. Matsuyama T. Kanetake H. J. Cell Sci... 2005; 118: 961-970Google Scholar). PEDF-deficient mice show increased stromal microvessel density in several organs, such as the pancreas and prostate, suggesting that PEDF plays a key role as a natural angiogenesis inhibitor (30Doll J.A. Stellmach V.M. Bouck N.P. Bergh A.R. Lee C. Abramson L.P. Cornwell M.L. Pins M.R. Borensztajn J. Crawford S.E. Nat. Med... 2003; 9: 774-780Google Scholar). PEDF activity is selective. It only targets new vessel growth and spares pre-existing vasculature. It seems that the anti-angiogenic effects of PEDF involve endothelial cell death through the activation of the Fas/FasL death pathway (31Volpert O.V. Zaichuk T. Zhou W. Reiher F. Ferguson T.A. Stuart P.M. Amin M. Bouck N.P. Nat. Med... 2002; 8: 349-357Google Scholar). MAPK JNK and p38 influence endothelial cell apoptosis by modulating c-FLIP or caspase activity in the presence of PEDF (32Zaichuk T.A. Shroff E.H. Emmanuel R. Filleur S. Nelius T. Volpert O.V. J. Exp. Med... 2004; 199: 1513-1522Google Scholar, 33Chen L. Zhang S.S. Barnstable C.J. Tombran-Tink J. Biochem. Biophys. Res. Commun... 2006; 348: 1288-1295Google Scholar). Recently, Cai et al. (34Cai J. Jiang W.G. Grant M.B. Boulton M. J. Biol. Chem... 2006; 281: 3604-3613Google Scholar) reported that PEDF inhibits VEGF-induced angiogenesis in retinal ECs. PEDF enhances γ-secretase-dependent cleavage of the C terminus of VEGF receptor-1, thus blocking VEGF receptor-2 induced angiogenesis. This study aimed to investigate potential receptors for PEDF and to establish how they influence angiogenesis. We used a yeast two-hybrid (Y2H) approach to identify potential PEDF partners, paying particular attention to proteins that could be PEDF receptors. Our results demonstrate that the non-integrin 37/67-kDa laminin receptor (LR) is a new PEDF receptor. LR could be the proposed 60-kDa receptor identified in ECs (24Yamagishi S. Inagaki Y. Nakamura K. Abe R. Shimizu T. Yoshimura A. Imaizumi T. J. Mol. Cell Cardiol... 2004; 37: 497-506Google Scholar). LR is not simply a laminin receptor. It also mediates prion protein internalization (35Gauczynski, S., Peyrin, J. M., Haïk, S., Leucht, C., Hundt, C., Rieger, R., Krasemann, S., Deslys, J. P., Dormont, D., Lasmézas, C. I., and Weiss, S. EMBO J. 20, 5863–5875Google Scholar) and functions as a receptor for viruses, such as Sindbis, dengue, and adeno-associated virus (36Wang K.S. Kuhn R.J. Strauss E.G. Ou S. Strauss J.H. J. Virol... 1992; 66: 4992-5001Google Scholar, 37Thepparit C. Smith D.R. J. Virol... 2004; 78: 12647-12656Google Scholar-38Akache B. Grimm D. Pandey K. Yant S.R. Xu H. Kay M.A. J. Virol... 2006; 80: 9831-9836Google Scholar). The LR subunit is implicated in several physiological and pathological processes, including cell differentiation, growth, migration, and cancer invasion (39Nelson J. McFerran N.V. Pivato G. Chambers E. Doherty C. Steele D. Timson D.J. Biosci. Rep... 2008; 28: 33-48Google Scholar). Our research shows that LR helps mediate PEDF anti-angiogenic activities. We identified both a 25-mer LR-interacting domain on PEDF and a PEDF-interacting domain on LR. The 25-mer PEDF-derived peptide exerts the same anti-angiogenic and pro-apoptotic effects on ECs as PEDF. Y2H Screening of PEDF Partners—We used a Matchmaker GAL4 two-hybrid system of Saccharomyces cerevisiae AH109 strain (Clontech) to screen a human skeletal muscle Matchmaker cDNA library (Clontech), with their 5′ ends proximal to the activation domain (AD) of the GAL4 transcription factor in a pACT2 vector. We used full-length PEDF cDNA (accession number NM_002615) baits, cloned in a pGBKT7 vector, with the GAL4-binding domain (BD) at their 5′ end. We performed interaction selection on high stringency medium (SD/–Ade/–His/–Leu/–Trp/X-α-Gal). The AD-containing plasmids in the selected clones were isolated according to the manufacturer's instructions. We determined the cDNA nucleotide sequences in each clone (genome-express, Meylan, France) and compared them with the GenBank™ data base by using the BLAST search program. Y2H Method to Identify Laminin Receptor and PEDF Interaction—We PCR-amplified different human PEDF fragments (encoding amino acids 2–418, 140–418, 206–418, 374–418, 2–326, 2–140, 2–86, 2–44, 44–121, and 44–77; see Fig. 1) and cloned them into the EcoRI and BamHI sites of the pGBKT7 vector. Similarly, we PCR-amplified LR fragments (encoding amino acids 2–295, 96–295, 96–295del158–179, 120–210, 135–200, 157–210, 120–180, and 157–180) with probes containing EcoRI and BamHI sites and cloned them into pGADT7. We verified the constructions by sequencing. We studied the potential interaction between different fragments of PEDF and LR by co-transforming them into the S. cerevisiae AH109 strain, as described above. Yeast immunofluorescence was performed as described (40Lee F.J. Huang C.F. Yu W.L. Buu L.M. Lin C.Y. Huang M.C. Moss J. Vaughan M. J. Biol. Chem... 1997; 272: 30998-31005Google Scholar) with anti-GAL4-AD and anti-GAL4-BD (Santa Cruz). Yeast protein extracts were carried out as described (41von der Haar T. PLoS ONE.. 2007; 2: e1078Google Scholar) and analyzed with anti-GAL4-AD and anti-LR (Santa Cruz) in Western blot analysis. Cell Cultures—Human bone marrow endothelial cells (HuBMEC) (42Schweitzer K.M. Vicart P. Delouis C. Paulin D. Dräger A.M. Langenhuijsen M.M. Weksler B.B. Lab. Investig... 1997; 76: 25-36Google Scholar) were grown in endothelial cell basal medium 2 (Promocell, Heidelberg, Germany). We cultured cells in plates coated with 0.2% gelatin (Sigma) at 37 °C in a humidified atmosphere of 5% CO2. We grew COS7 cells in Dulbecco's modified Eagle's medium, supplemented with 10% FBS with 100 units/ml penicillin and 100 μg/ml streptomycin. Expression Vector Construction and Transfection into Cell Cultures—We cloned full-length PEDF (aa 2–418) and LR (aa 2–295) cDNAs in pCMV-HA and pCMV-Myc vectors (Clontech) to obtain pCMV-HA-tagged-PEDF (HA-PEDF) and Myc-tagged-LR (Myc-LR) expression vectors, respectively. We then co-transfected these two constructions into COS-7 cells that we had plated at 1.5 × 105 cells/well the previous day using Matra transfection reagent, according to the manufacturer's instructions (IBA, Göttingen, Germany). PEDF Expression in Insect Cells and Laminin Receptor Expression in Escherichia coli—We used the primers PEDF2 (5′-AATGAATTCCAGGCCCTGGTGCTACTCCTC-3′) and PEDFR (5′-CCTCTAGACTGGGGCCCCTGGGGTCCAG-3′) to PCR amplify human PEDF cDNA (2–418). We cloned this fragment into the EcoRI and XbaI sites of the pIB/V5-His/CAT vector (Invitrogen). Primers LR2 (5′-ACTGAATTCTCCGGAGCCCTTGATGTCCTG-3′) and LR reverse primer (5′-ACTGCGGCCGCAGACCAGTCAGTGGTTGCTCC-3′) or LR90 (5′-ACTGAATTCGCCACTCCAATTGCTGGCCGC-3′) and LRR were used to PCR amplify human LR (2–295) or LR90 (90–295) fragments, respectively. We cloned these fragments into the EcoRI and NotI sites of the pSCodon2 vector (Delphi Genetics, Charleroi, Belgium). We purified PEDF from Sf9 insect cells after blasticidin selection and purified laminin receptor from SE1 bacteria after isopropyl β-d-thiogalactopyranoside induction with Ni-NTA-agarose resin (Qiagen), according to the manufacturer's instructions. Immunofluorescence—COS-7 cells were co-transfected with HA-PEDF and Myc-LR. Forty-eight hours later, we rinsed them with PBS twice and fixed them in 4% paraformaldehyde for 5 min, followed by 50 mm NH4Cl for 15 min. We permeabilized cells with PBS containing 0.1% Triton X-100 for 10 min. We then incubated the permeabilized cells with PBS containing 2% bovine serum albumin for 20 min before incubating them with either anti-PEDF antibody (MAB1059, 1:100; Chemichon, Temecula, CA) and anti-LR antibody (H-141, 1:100; Santa Cruz) or anti-HA antibody (3F10, 1:100; Roche Applied Science) and anti-Myc tag antibody (9B11, 1:2000; Cell Signaling, Beverly, MA) in PBS containing 0.2% bovine serum albumin for 1 h. We washed cells four times with PBS and incubated them with Cy3-conjugated Goat anti-rabbit antibody (1:400, Jackson Immunoresearch; Suffolk, UK) or Alexa Fluor 488 goat anti-mouse IgG (1:400; Molecular Probes, Carlsbad, CA) for 1 h in the dark. We washed the cells three times in PBS before incubating them with DAPI (dilution 1:10000, Sigma) for 5 min. We washed cells twice before mounting them onto glass slides with a drop of Mowiol mounting medium. Molecular Modeling—We modeled the PEDF-interacting domain using VMD software. We reduced the region into a theoretical functional domain with the help of a server and a docking server simulating an interaction between the peptide and ribosomal protein S2, which shares a 52% sequence homology with LR. One peptide, 25-mer P46 (aa 46–70, FFKVPVNKLAAAVSNFGYDLYRVRS) was determined and synthesized (see Fig. 3). We also synthesized fluoroscein-coupled peptide (F46) (Engineering protein platform, IFR83, UPMC, Paris, France). Surface Plasmon Resonance (SPR) Assays—Using a BIAcore 3000 instrument (Biacore AB, Uppsala, Sweden), we analyzed the molecular interaction in real time between PEDF or PEDF-derived peptide P46 and his-LR (aa 2–295) or his-LR90 (aa 90–295) isolated from E. coli and immobilized on a NTA sensor chip to reach a response of between 1200 and 1600 resonance units (RU), according to the manufacturer's recommendations. We prepared a reference surface, without protein, by the same procedure. To evaluate nonspecific background signals, we also used PEDF protein and peptide P46 on NTA chips uncoated with His-LR. Running buffer (10 mm HEPES, 150 mm NaCl, 50 μm EDTA, 0.005% surfactant P20, pH 7.4) and binding buffer (10 mm HEPES, 150 mm NaCl, 5 mm CaCl2, 50 μm MgCl2, 50 μm EDTA, 0.005% surfactant P20, pH 7.4) were used. We used PEDF protein (reference number 01-211; Upstate) and peptide P46 to study binding. We analyzed results with BIA evaluation software version 4.1. F46 Binding to Cells—We grew HuBMECs to a 60–80% confluency before incubating them with fluoroscein-coupled-P46 peptide (F46, 50 or 200 nm) at 4 °C for 60 min. To test the binding specificity, we used either nonfluorescent peptide (P46) at 500 μm or anti-LR antibodies in the binding assay. In the latter case, HuBMECs were first preincubated for 30 min with LR antibody (67LR, ab711, 1:100; Abcam, Cambridge, UK; or LR antibody, H-141, 1:100; Santa Cruz) at 37 °C, followed by placing the plate at 4 °C for 30 min, prior to incubation with F46 (50 or 200 nm) at 4 °C for 1 h. We washed cells three times with PBS and then fixed them with 4% paraformaldehyde for 5 min, incubated them with PBS containing DAPI, then washed, and mounted them beneath glass coverslips. We took photographs with a Zeiss LSM510 confocal laser microscope (Göttingen, Germany). We used a nonrelevant rhodamin-labeled 25-mer peptide KAP3.1 (FSDKSCRCGVCLPSTCPHEISLLQP) derived from keratin-associated protein (43Thibaut S. Cavusoglu N. De Becker E. Zerbib F. Bednarczyk A. Schaeffer C. Van Dorsselaer A. Bernard B.A. J. Investig. Dermatol... 2008; 129: 449-459Google Scholar) as a control peptide. Western Blot Analysis—We extracted proteins using radioimmune precipitation assay buffer (1 mm EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 150 mm NaCl, 50 mm Tris-HCl, pH 7.25, 1 mm phenylmethylsulfonyl fluoride, and a mixture of protease inhibitors (Roche Applied Science)). We determined protein amounts using Bradford reagent (BioRad). We denatured 20 μg of protein with reducing Laemmli buffer and resolved them with 12% SDS-PAGE. We then transferred the proteins to the polyvinylidene difluoride membranes (Amersham Biosciences). We blocked the membranes with 5% fatty acid-free milk at room temperature for 1 h. We then incubated the membranes overnight at 4 °C, with the primary antibodies (LR antibody, H-141, 1:250; Santa Cruz; and active caspase-3, CPP32, 1:1000; R & D System) diluted in 5% milk, followed by incubation with a secondary antibody conjugated with peroxidase (Dako, Glostrup, Denmark). We used ECL detection reagent to detect signals. The values obtained with ImageJ software were normalized with anti-glyceraldehyde-3-phosphate dehydrogenase antibody (FL-335, 1:400; Santa Cruz). Co-immunoprecipitation—We incubated the protein mixtures at 4 °C for 1 h with 2 μg of anti-PEDF antibody (Chemicon) on a mixing rotor. We added agarose A/G protein (20 μl) and incubated the samples overnight. The next day, we centrifuged the samples at 1000 × g for 5 min. We removed the supernatant, leaving a pellet of beads. We washed the pellets four times by centrifuge (1000 × g, 5 min) with radioimmune precipitation assay buffer (1 ml) (1× PBS, pH 7.4, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), adding Laemmli buffer after the last wash. We performed Western blot analysis with anti-LR antibody (H-141, 1:250; Santa Cruz) as described above. His Tag Pulldown Assay—We assessed PEDF binding to His-tagged LR by His tag pulldown of bound complexes with Ni-NTA resin, as described by Notari et al. (26Notari L. Baladron V. Aroca-Aguilar J.D. Balko N. Heredia R. Meyer C. Notario P.M. Saravanamuthu S. Nueda M.L. Sanchez-Sanchez F. Escribano J. Laborda J. Becerra S.P. J. Biol. Chem... 2006; 281: 38022-38037Google Scholar). PEDF protein (2 μg; Upstate) was mixed with either 1 μg of His-LR (aa 2–295) or 1 μg of His-LR90 (aa 90–295, extracellular domain), purified from E. coli by Ni-NTA resin (more than 95% purity) in binding buffer (50 mm sodium phosphate, pH 7.5, 500 mm Nacl, 1% Nonidet P-40; final volume, 150 μl), and incubated at 4 °C for 4 h with gentle rotation. We added the Ni-NTA resin beads (50 μl), pre-equilibrated in binding buffer, to the mixture and incubated at 4 °C for 2 h with gentle rotation. Brief centrifugation sedimented the resin beads, and we washed them three times with binding buffer. We extracted the proteins with 50 μl of 2× Laemmli buffer and analyzed them by Western blot with anti-PEDF antibody. Matrigel Angiogenesis Assay—We performed this assay as previously described (44Franco C.A. Mericskay M. Parlakian A. Gary-Bobo G. Gao-Li J. Paulin, D. Gustafsson E. Li Z. Dev. Cell.. 2008; 15: 448-461Google Scholar). HuBMECs were seeded in 24-well plates, precoated with 300 μl of growth factor-reduced Matrigel (BD Bioscience), at 48,000 cells/well. We cultured cells for 24 h in Dulbecco's modified Eagle's medium, supplemented with 0.2% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. We examined the effects of PEDF, 25-mer peptide P46, or control peptide KAP3.1 on bFGF-induced tube-like networks by phase contrast microscopy. We took representative photographs from four random fields/sample and measured the endothelial tube lengths. We then performed three distinct experiments. Corneal Angiogenesis—We assessed corneal angiogenesis as described previously (45Kenyon B.M. Voest E.E. Chen C.C. Flynn E. Folkman J. D'Amato R.J. Investig. Ophthalmol. Vis. Sci... 1996; 37: 1625-1632Google Scholar). Sucralfate pellets containing PBS alone, bFGF alone, bFGF plus PEDF, bFGF plus peptide P46, or P46 alone were implanted into the corneas of C57/B16 mice. We used about 30 ng of bFGF, 50 ng of PEDF, and 10 ng of P46/pellet. Three to eight animals (6–11 corneal implants) were examined per sample. We used an Olympus SCH10 microscope to examine the eyes of the mice. The Pierre and Marie Curie University Animal Care and Use Committee had approved the protocol. Apoptosis Assay—We performed terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end labeling (TUNEL) using an In Situ Cell Death Detection Kit (Roche Applied Science). We seeded HuBMECs at 3.8 × 104 cells/well in 24-well plates in complete MEB2 medium (Promocell). The next day the cells were serum-starved for 14 h by incubation in 0.2% serum MEB2 without growth factors. We then incubated cells with PEDF (40 ng/ml), P46 (200 nm), or KAP3.1 (200 nm) in the presence or absence of bFGF (20 ng/ml) and VEGF (20 ng/ml) for 24 h. We rinsed cells with PBS (pH 7.4) for 5 min twice, fixed them with 4% paraformaldehyde, and stained them according to the manufacturer's instructions. We stained cell nuclei with DAPI. We assessed the percentage of TUNEL-stained cells by fluorescence microscopy, examining four random views/sample. We repeated the experiments three times. Wound Healing Assay—We plated HuBMECs into 12-well plates at 50,000 cells/cm2 and grew them until confluent. We created a wound using a pipette cone. We then washed cells once with culture medium and left them in Dulbecco's modified Eagle's medium supplemented with 0.5% FBS and 20 ng/ml bFGF. We monitored the wounded areas over 24 h and took micrographs every 4 h. siRNA-mediated LR Knockdown Experiment—We seeded HuBMECs in 6-well dishes at 1 × 105 cells/well 1 day before transfection. We used four siRNA oligonucleotides (Genecust, Ivry, France) to target the human LR gene, either individually or in combination: LR-100 (5′-GGAACAGUACAUCUAUAAATT-3′/5′-UUUAUAGAUGUACUGUUCCAT-3′), LR-211 (5′-UGCUGAUGUCAGUGUUAUATT-3′/5′-UAUAACACUGACAUCAGCAGG-3′), LR-402 (5′-CGGAGGCAUCUUAUGUUAATT-3′/5′-UUAACAUAAGAUGCCUCCGTG-3′), and LR-612 (5′-CAGAGAUCCUGAAGAGAUUTT-3′/5′-AAUCUCUUCAGGAUCUCUGTT-3′). We used scrambled siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′/5′-ACGUGACACGUUCGGAGAATT-3′) as a negative control. The siRNA pool of the four sequences gave the best knockdown result (data not shown), and we used this in all subsequent experiments. We transfected cells with MATRA-si (IBA) 24 h later, according to the manufacturer's protocol. We analyzed silencing efficiency by immunoblotting with anti-LR antibody (H-141; Santa Cruz). We assayed active caspase-3 levels by using anti-active caspase-3 antibody (CPP32; R & D System) in a Western blot. We repeated the experiments twice. Statistical Analysis—We analyzed quantitative data using Fisher post hoc tests for repeated measures and Student's unpaired t tests. The data shown are the means ± S.E. We considered p values <0.05 to be statistically significant and have marked them with asterisks. Identification of 37/67-kDa LR as a PEDF Partner by the Y2H System—Considering the widespread tissue expression and distinct biological functions of PEDF, we assumed that this peptide would have a series of partners. To investigate this further, we adopted a Y2H approach, screening a human skeletal muscle cDNA library using full-length PEDF (aa 2–418) as bait. We screened the clones grown on high stringency selected medium (SD/–Ade/–His/–Leu/–Trp/X-α-Gal). We isolated and sequenced plasmids from the clones that expressed proteins potentially interacting with GAL4-BD-PEDF. We then matched the sequences from these clones with the GenBank™ data base. Sequence analysis revealed that several of these clones encode plasma membrane proteins or potential PEDF receptors, such as PNPLA2 and 37/67-kDa LR. During our study, Becerra and co-workers (26Notari L. Baladron V. Aroca-Aguilar J.D. Balko N. Heredia R. Meyer C. Notario P.M. Saravanamuthu S. Nueda M.L. Sanchez-Sanchez F. Escribano J. Laborda J. Becerra S.P. J. Biol. Chem... 2006; 281: 38022-38037Google Scholar) identified PNPLA2 as a PEDF receptor, one with potent phospholipase A2 activity that liberates fatty acids. We therefore decided to focus on the secon

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