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• W2076304369 abstract "ADAMTS1 is an extracellular metalloproteinase known to participate in a variety of biological processes that includes inflammation, angiogenesis, and development of the urogenital system. Many of its functions rely on its catalytic activity, which thus far has been limited to the cleavage of the matrix proteoglycans aggrecan and versican. However, it is likely that other substrates exist. Using a yeast two-hybrid screen, we identified the Kunitz-type inhibitor, tissue factor pathway inhibitor-2 (TFPI-2), as a binding partner of ADAMTS1. The interaction was confirmed by several biochemical and cell-based assays. In addition, our studies revealed alterations in the pattern of TFPI-2-secreted isoforms and in its extracellular location caused by the specific action of ADAMTS1. Interestingly, we found that TFPI-2 is a novel substrate of ADAMTS1. The cleavage removes a protease-sensitive C-terminal region in TFPI-2, altering its binding properties. The proposed role of TFPI-2 as a maintenance factor of extracellular remodeling suggests the indirect function of ADAMTS1 as an additional homeostatic player by its ability to alter the extracellular location of TFPI-2 and, therefore, to disrupt the remodeling machinery, a phenomenon directly associated to pathologies such as atherosclerosis and tumor progression. ADAMTS1 is an extracellular metalloproteinase known to participate in a variety of biological processes that includes inflammation, angiogenesis, and development of the urogenital system. Many of its functions rely on its catalytic activity, which thus far has been limited to the cleavage of the matrix proteoglycans aggrecan and versican. However, it is likely that other substrates exist. Using a yeast two-hybrid screen, we identified the Kunitz-type inhibitor, tissue factor pathway inhibitor-2 (TFPI-2), as a binding partner of ADAMTS1. The interaction was confirmed by several biochemical and cell-based assays. In addition, our studies revealed alterations in the pattern of TFPI-2-secreted isoforms and in its extracellular location caused by the specific action of ADAMTS1. Interestingly, we found that TFPI-2 is a novel substrate of ADAMTS1. The cleavage removes a protease-sensitive C-terminal region in TFPI-2, altering its binding properties. The proposed role of TFPI-2 as a maintenance factor of extracellular remodeling suggests the indirect function of ADAMTS1 as an additional homeostatic player by its ability to alter the extracellular location of TFPI-2 and, therefore, to disrupt the remodeling machinery, a phenomenon directly associated to pathologies such as atherosclerosis and tumor progression. The extracellular milieu has been recognized as a dynamic scenario that directly influences proliferation, survival, migration, and biosynthetic activities of cells. The constituents of this extracellular environment include growth factors, chemokines, cell surface proteins, and an extensive list of matrix components with structural and signaling properties. Matrix proteases and their respective inhibitors are also important components of the extracellular milieu with the added value that these molecules modify, degrade, or inflict functional alterations to most of the components previously listed (1Handsley M.M. Edwards D.R. Int. J. Cancer. 2005; 115: 849-860Crossref PubMed Scopus (236) Google Scholar). In fact, modification of extracellular components by processing and/or proteolysis is a frequent event during morphogenesis and tissue repair and, in an altered manner, during the progression of various pathological conditions (2Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases includes a group of 19 secreted enzymes with a more restricted spectrum of catalytic activities than the well known matrix metalloproteinases (MMP) 2The abbreviations used are: MMP, matrix metalloproteinase; ADAM, a disintegrin and metalloproteinase domain; ADAMTS, a disintegrin-like and metalloprotease with thrombospondin type I motifs; ECM, extracellular matrix; PMA, phorbol 12-myristate 13-acetate; TFPI-2, tissue factor (TF) pathway inhibitor 2; TSR, thrombospondin type I repeat; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; pAb, polyclonal antibody; mAb, monoclonal antibody; RIPA, radioimmune precipitation assay. 2The abbreviations used are: MMP, matrix metalloproteinase; ADAM, a disintegrin and metalloproteinase domain; ADAMTS, a disintegrin-like and metalloprotease with thrombospondin type I motifs; ECM, extracellular matrix; PMA, phorbol 12-myristate 13-acetate; TFPI-2, tissue factor (TF) pathway inhibitor 2; TSR, thrombospondin type I repeat; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; pAb, polyclonal antibody; mAb, monoclonal antibody; RIPA, radioimmune precipitation assay. (3Porter S. Clark I.M. Kevorkian L. Edwards D.R. Biochem. J. 2005; 386: 15-27Crossref PubMed Scopus (617) Google Scholar). ADAMTS1, the first member described (4Kuno K. Kanada N. Nakashima E. Fujiki F. Ichimura F. Matsushima K. J. Biol. Chem. 1997; 272: 556-562Abstract Full Text Full Text PDF PubMed Scopus (437) Google Scholar), has been shown to display anti-angiogenic properties (5Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar). Systemic deletion of adamts1 in mice resulted in multiple defects in the urogenital system (6Shindo T. Kurihara H. Kuno K. Yokoyama H. Wada T. Kurihara Y. Imai T. Wang Y. Ogata M. Nishimatsu H. Moriyama N. Oh-hashi Y. Morita H. Ishikawa T. Nagai R. Yazaki Y. Matsushima K. J. Clin. Investig. 2000; 105: 1345-1352Crossref PubMed Scopus (272) Google Scholar, 7Mittaz L. Russell D.L. Wilson T. Brasted M. Tkalcevic J. Salamonsen L.A. Hertzog P.J. Pritchard M.A. Biol. Reprod. 2004; 70: 1096-1105Crossref PubMed Scopus (146) Google Scholar). The enzyme is known to have catalytic activity toward matrix proteoglycans, particularly versican and aggrecan (8Sandy J.D. Westling J. Kenagy R.D. Iruela-Arispe M.L. Verscharen C. Rodriguez-Mazaneque J.C. Zimmermann D.R. Lemire J.M. Fischer J.W. Wight T.N. Clowes A.W. J. Biol. Chem. 2001; 276: 13372-13378Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar, 9Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (205) Google Scholar). Mechanistically, it is not evident that the outcomes of the gene-deletion studies can be linked with the catalytic profile known for this enzyme. Thus, it is possible that either other domains contribute to the spectrum of biological functions and/or that additional biologically relevant substrates are yet to be identified. The modular structure of ADAMTS1 allows for a multiplicity of protein-protein interactions that are likely to affect its biological actions. Here, we have exploited the yeast two-hybrid system to identify potential interacting proteins. The screen resulted in the identification of tissue factor pathway inhibitor-2 (TFPI-2) as a binding partner for ADAMTS1. TFPI-2 was first identified as a serine proteinase inhibitor containing Kunitz domains (10Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Crossref PubMed Scopus (185) Google Scholar, 11Miyagi Y. Koshikawa N. Yasumitsu H. Miyagi E. Hirahara F. Aoki I. Misugi K. Umeda M. Miyazaki K. J Biochem. (Tokyo). 1994; 116: 939-942Crossref PubMed Scopus (95) Google Scholar). Its inhibitory abilities have been demonstrated toward tissue factor (TF)-Factor VIIa complex, trypsin, plasmin, and kallikrein (12Petersen L.C. Sprecher C.A. Foster D.C. Blumberg H. Hamamoto T. Kisiel W. Biochemistry. 1996; 35: 266-272Crossref PubMed Scopus (139) Google Scholar). Consequently, this inhibitor affects the plasmin- and trypsin-mediated activation of matrix metalloproteinases proMMP-1 and proMMP-3 (13Rao C.N. Mohanam S. Puppala A. Rao J.S. Biochem. Biophys. Res. Commun. 1999; 255: 94-98Crossref PubMed Scopus (87) Google Scholar). In addition, direct inhibition of MMP1, -2, -9, and -13 activities have been reported (14Herman M.P. Sukhova G.K. Kisiel W. Foster D. Kehry M.R. Libby P. Schonbeck U. J. Clin. Investig. 2001; 107: 1117-1126Crossref PubMed Scopus (195) Google Scholar). TFPI-2 is secreted by several cell types, including endothelial cells (15Rao C.N. Gomez D.E. Woodley D.T. Thorgeirsson U.P. Arch. Biochem. Biophys. 1995; 319: 55-62Crossref PubMed Scopus (37) Google Scholar), smooth muscle cells, fibroblasts (16Rao C.N. Liu Y.Y. Peavey C.L. Woodley D.T. Arch. Biochem. Biophys. 1995; 317: 311-314Crossref PubMed Scopus (65) Google Scholar), keratinocytes (17Rao C.N. Peavey C.L. Liu Y.Y. Lapiere J.C. Woodley D.T. J. Investig. Dermatol. 1995; 104: 379-383Abstract Full Text PDF PubMed Scopus (35) Google Scholar), and syncytiotrophoblasts (18Udagawa K. Miyagi Y. Hirahara F. Miyagi E. Nagashima Y. Minaguchi H. Misugi K. Yasumitsu H. Miyazaki K. Placenta. 1998; 19: 217-223Crossref PubMed Scopus (55) Google Scholar). Its preferential association to the extracellular matrix (ECM) has been documented (19Liu Y. Stack S.M. Lakka S.S. Khan A.J. Woodley D.T. Rao J.S. Rao C.N. Arch. Biochem. Biophys. 1999; 370: 112-118Crossref PubMed Scopus (33) Google Scholar), and several reports suggest a pivotal role of TFPI-2 in the maintenance and regulation of ECM remodeling. In fact, altered levels of TFPI-2 have been associated with pathological conditions, such as atherosclerosis (14Herman M.P. Sukhova G.K. Kisiel W. Foster D. Kehry M.R. Libby P. Schonbeck U. J. Clin. Investig. 2001; 107: 1117-1126Crossref PubMed Scopus (195) Google Scholar) and tumor growth, although its action in this second case appears paradoxical and dependent on the tumor model studied. For example, its expected inhibitory role was observed in models of glioma invasion (20Konduri S.D. Rao C.N. Chandrasekar N. Tasiou A. Mohanam S. Kin Y. Lakka S.S. Dinh D. Olivero W.C. Gujrati M. Foster D.C. Kisiel W. Rao J.S. Oncogene. 2001; 20: 6938-6945Crossref PubMed Scopus (72) Google Scholar) and fibrosarcoma growth and metastasis (21Chand H.S. Du X. Ma D. Inzunza H.D. Kamei S. Foster D. Brodie S. Kisiel W. Blood. 2004; 103: 1069-1077Crossref PubMed Scopus (67) Google Scholar). In contrast, TFPI-2 exerts a pro-invasive effect on hepatocarcinoma (22Neaud V. Hisaka T. Monvoisin A. Bedin C. Balabaud C. Foster D.C. Desmouliere A. Kisiel W. Rosenbaum J. J. Biol. Chem. 2000; 275: 35565-35569Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In aggressive uveal melanoma, this molecule was found to be up-regulated when compared with poorly aggressive melanoma (23Ruf W. Seftor E.A. Petrovan R.J. Weiss R.M. Gruman L.M. Margaryan N.V. Seftor R.E. Miyagi Y. Hendrix M.J. Cancer Res. 2003; 63: 5381-5389PubMed Google Scholar). Interestingly, in this latter study, TFPI-2 was implicated in processes of vasculogenic mimicry, an alternative pathway for tumor perfusion that is independent of angiogenesis (for review, see Ref. 24Folberg R. Maniotis A.J. APMIS. 2004; 112: 508-525Crossref PubMed Scopus (250) Google Scholar). In this study we confirmed the interaction between ADAMTS1 and TFPI-2 by several independent approaches. Moreover, our results indicate that the interaction regulates the extracellular distribution of TFPI-2 through the proteolytic processing at its C-terminal end. These effects were observed in both cell culture and tumor xenograft assays. Taken together, these findings suggest that ADAMTS1 is a regulator of TFPI-2 function by modulating its distribution within the extracellular microenvironment, implicating a role for ADAMTS1 in pathological processes that includes, but is not restricted to, tumor progression and atherosclerosis. Yeast Two-hybrid Screening—The MATCHMAKER Two-hybrid System 2 (Clontech Laboratories Inc.) was used according to the manufacturer. A cDNA fragment encoding amino acid residues 540–666 of human ADAMTS1 was amplified by PCR and inserted into the pAS2–1 vector that was previously digested with NdeI and SalI. The resulting GAL-4BD-ADAMTS1540–666 chimera protein was used as bait to screen a human placenta MATCHMAKER cDNA library (Clontech). Both bait and library vectors were simultaneously introduced into Saccharomyces cerevisiae CG-1945, and double transformants were selected on synthetic minimal medium with dextrose but lacked tryptophan, leucine, and histidine. A total of ∼7 × 105 clones was screened. Putative positive colonies were re-streaked and tested for β-galactosidase activity using a filter assay. DNA fragments from positive colonies were amplified by PCR using the MATCHMAKER 5′- and 3′-AD LD-Insert Screening Amplimers and then sequenced. Expression Vectors—Bicistronic TFPI-2-green fluorescent protein expression vector was obtained as follows. pZem 229 plasmid (10Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Crossref PubMed Scopus (185) Google Scholar) containing the TFPI-2 cDNA was digested with EcoRI and subsequently cloned into the EcoRI pIRES2-EGFP (BD Biosciences Clontech). To generate the TFPI-2-Myc expression vector (scheme in Fig. 1B), pZem229 was used as a template to amplify and create new restriction sites with the following oligonucleotides: TFPI-2 forward 5′-C AGA ATT CCC ATG GAC CCC GCT CGC-3′, TFPI-2 reverse 5′-CT TGG TAC CTG CTT CTT CCG AAT TTT C-3′ (EcoRI and KpnI restriction sites are in bold, and the starting ATG is underlined). The resulting PCR product was then cloned into pCR2.1 (Invitrogen) using the TOPO-TA cloning kit, and the TFPI-2 cDNA was obtained by subsequent digestion with EcoRI and KpnI. This fragment was inserted into KpnI/EcoRI pcDNA 3.1/Myc-His(–) B expression vector (Invitrogen). This construct contains a functional Myc-His epitope at the C-terminal end. The C-terminal-truncated TFPI-2 (ΔCTFPI-2-Myc) expression vector (scheme in Fig. 1B) was generated by amplification of pZem229 with the same forward primer noted above and the reverse primer 5′-CTT GGT ACC CTT TTT CAA AGC TTT TGC-3′. The same procedure described above was used to subclone this truncated form into KpnI/ EcoRI pcDNA 3.1/Myc-His(–) B expression vector (Invitrogen). Full-length human ADAMTS1, zinc-binding site mutant E385A-ADAMTS1, and ΔTSRs-ADAMTS1 (Met-Dis: 1–556) constructs have previously been described (9Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (205) Google Scholar, 25Luque A. Carpizo D.R. Iruela-Arispe M.L. J. Biol. Chem. 2003; 278: 23656-23665Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Recombinant human TFPI-2 and p87-ADAMTS1 were purified as described (10Sprecher C.A. Kisiel W. Mathewes S. Foster D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3353-3357Crossref PubMed Scopus (185) Google Scholar, 26Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Cell Culture, Transfection, and Treatments—Mouse lung endothelial (LE) and 293T cells were cultured at 37 °C with 5% CO2 under saturated humidity in Dulbecco's modified Eagle's medium (PAA Laboratories, Pasching, Austria) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin-streptomycin, l-glutamine, and plasmocin. Transient and stable transfections were performed with FuGENE 6 transfection reagent (Roche Diagnostics) according to the manufacturer's specification. pLSHL-Hygro vector (Invitrogen) was co-transfected in stable transfection experiments for further selection with hygromycin. Additional stable cell lines used in this study are 293T overexpressors of ADAMTS1 (26Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar) and zinc-binding site mutant E385A-ADAMTS1 (9Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (205) Google Scholar). Conditioned medium from stable 293T cell lines was collected after 48 h of incubation in serum-free medium. For transient transfection experiments, cells were maintained for 24 h in 10% fetal calf serum and then switched to serum-free medium before collecting the conditioned medium and harvesting the cell lysates. Cell culture treatments are as follows: 5 μg/ml heparin (Sigma-Aldrich), 1 μm phorbol 12-myristate 13-acetate (PMA) (Calbiochem), 1 μg/ml tunicamycin (Sigma-Aldrich), 6 μm protease inhibitor BB94 (a generous gift from Richard Kenagy, University of Washington, Seattle, WA), 5 milliunits/ml plasmin (Roche Applied Science), 1 units/ml thrombin (Sigma), and 2 μg/ml aprotinin (Sigma), 20 μg/ml leupeptin (Sigma). In all cases treatments were performed in serum-free media. For lung endothelial treatments, cultures were serum-deprived overnight before treatment with the indicated conditioned medium or recombinant proteins diluted in serum-free Dulbecco's modified Eagle's medium. Conditioned medium was collected, and the cell layer was rinsed twice with PBS and harvested in radioimmune precipitation assay (RIPA) buffer (20 mm Tris-HCl, pH 8, 150 mm NaCl, 0.1% SDS, 1% Nonidet P-40, 1 mm EDTA). N-Glycosidase F Treatment—Conditioned medium was methanolprecipitated and re-suspended in 50 μl of reaction buffer (100 mm sodium phosphate, pH 7.0, 2% SDS, 0.1 mm EDTA). Samples were incubated for 3 min at 100 °C, and then 0.1% Triton X-100 was added. Enzymatic incubation was performed at 37 °C for 20 h in the presence of 1 unit of N-glycosidase F (Roche Applied Science). In Vitro Digestion of TFPI-2—Conditioned medium from stable TFPI-2-Myc overexpressing 293T cells was purified under denaturing conditions (PBS, pH 8, 8 m urea) with nickel nitrilotriacetic acid-agarose (Qiagen). Bound protein was washed with PBS and equilibrated with reaction buffer (20 mm Tris, pH 7.4, 100 mm NaCl, 10 mm CaCl2, 10 μm ZnCl2, 5 μg/ml heparin). Digestions were performed at 37 °C for 16 h. Then samples were concentrated with StrataClean resin (Stratagene, Cedar Creek, TX), resolved by SDS-PAGE, and analyzed by Western blot with Myc and TFPI-2 antibodies. Co-immunoprecipitation Studies—Cell lysates or conditioned media were preabsorbed with protein G-Sepharose 4 Fast Flow (Amersham Biosciences) for 30 min at 4 °C in a rotation wheel. This material was then incubated with the appropriate antibody overnight at 4 °C. Protein G-Sepharose was added for 1 h in the same condition. Finally, immunoprecipitates were pelleted, rinsed 3 times with RIPA/PBS (1:3), resuspended in loading buffer, and analyzed by SDS-PAGE. The antibodies used were monoclonal anti-ADAMTS1-clone 3E4C6B4 (9Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (205) Google Scholar) and monoclonal anti-Myc (a generous gift from Dr. Arribas, Vall d'Hebron University Hospital Research Institute, Barcelona, Spain). Tumor Xenograft Assays—293T clones were trypsinized, washed twice, and re-suspended in serum-free Dulbecco's modified Eagle's medium. Nu/Nu Balb/c mice were subcutaneously injected in the right back with 5 × 106 cells/200 μl. Mice weight and tumor size were assessed every 3 days after cell injection. When the tumors reached 1.5 cm2, mice were sacrificed, and the tumors were removed for further analysis. Tumor volumes were calculated by the equation D × d2 × π/6, where D is the tumor diameter at its widest, and d is at its smallest (27Matar P. Rojo F. Cassia R. Moreno-Bueno G. Di Cosimo S. Tabernero J. Guzman M. Rodriguez S. Arribas J. Palacios J. Baselga J. Clin. Cancer Res. 2004; 10: 6487-6501Crossref PubMed Scopus (274) Google Scholar). Heparin Purification—The affinity of ADAMTS1 and TFPI-2 proteins to heparin has been previously reported (19Liu Y. Stack S.M. Lakka S.S. Khan A.J. Woodley D.T. Rao J.S. Rao C.N. Arch. Biochem. Biophys. 1999; 370: 112-118Crossref PubMed Scopus (33) Google Scholar, 26Rodriguez-Manzaneque J.C. Milchanowski A.B. Dufour E.K. Leduc R. Iruela-Arispe M.L. J. Biol. Chem. 2000; 275: 33471-33479Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Samples were incubated with pre-equilibrated heparin beads (Amersham Biosciences) overnight at 4 °C under constant agitation. Heparin beads were then washed twice with 100 mm NaCl and twice more with PBS. Finally, samples were pelleted, resuspended in loading buffer, and analyzed by SDS-PAGE. Immunoblot Analysis—Cell layers and tumor samples were homogenized and lysed with RIPA buffer containing 1 mm of phenylmethylsulfonyl fluoride and 1 μg/ml aprotinin. Cell debris was discarded by centrifugation (20 min at 16,000 × g), and protein suspension was quantified using the Bio-Rad DC protein assay (Bio-Rad). When needed cell lysates were precipitated with 3 volumes of methanol at –20 °C overnight. The precipitate was washed twice with acetone and left to dry at room temperature. Conditioned medium was concentrated with Strata-Clean resin. Samples were subjected to SDS-PAGE or Tricine-SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked with 5% nonfat milk and incubated with the antibodies polyclonal rabbit anti-TFPI-2 (28Iino M. Foster D.C. Kisiel W. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 40-46Crossref PubMed Scopus (93) Google Scholar) and anti-TFPI (29Werling R.W. Zacharski L.R. Kisiel W. Bajaj S.P. Memoli V.A. Rousseau S.M. Thromb. Haemostasis. 1993; 69: 366-369Crossref PubMed Scopus (132) Google Scholar), polyclonal guinea pig anti-ADAMTS1 (5Vazquez F. Hastings G. Ortega M.A. Lane T.F. Oikemus S. Lombardo M. Iruela-Arispe M.L. J. Biol. Chem. 1999; 274: 23349-23357Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar), monoclonal anti-Myc (a generous gift from Dr. Arribas), and monoclonal anti-actin (clone AC-40, Sigma). After incubation with the appropriate secondary antibodies conjugated to peroxidase, signal was detected with the Super-Signal chemiluminescence kit (Pierce). Membranes were stripped as needed. Briefly, membranes were incubated with stripping buffer (62.5 mm Tris, pH 6.8, 2% SDS, and 100 mm β2-mercaptoethanol) for 30 min at 37 °C and then washed extensively. RNA Extraction and Reverse Transcription-PCR—Tumor samples were disrupted and homogenized in liquid nitrogen with mortar and pestle. Total RNA was extracted using the RNeasy Mini kit (Qiagen) and quantified in a NanoDrop ND-100 apparatus (NanoDrop Technologies, Wilmington, DE). Reverse transcription-PCR was performed with 0.5 μg of total RNA using oligo(dT) primers and the SuperScript First-Strand Synthesis system (Invitrogen) followed by specific amplification of TFPI-2 with the primers 5′-GGGCCCTACTTCTCCGTTAC (forward) and 5′-CACTGGTCGTCCACACTCAC (reverse). As an internal control, actin cDNA was amplified using the primers 5′-CCTGACCGAGCGTGGC TAC (forward) and 5′-GAAGCATTTGCGGTGGACG (reverse). Identification of TFPI-2 as an Interacting Protein of ADAMTS1—A yeast two-hybrid screen of a human placenta cDNA library was performed to identify ADAMTS1 interacting proteins. Given the multidomain structure of ADAMTS1 and the high probability of false positive clones, we decided to perform the screen using discrete modular domains of the coding region. A fragment of human ADAMTS1 containing the first thrombospondin type I repeat (TSR) and part of the cysteine-rich region (encoding amino acid residues 540–666) was used as bait (Fig. 1A). Among several positive clones identified, the sequencing of the clone F2, 3.3 included an insert that corresponded to the complete 3′-end (from nucleotide 258) of the human tissue factor pathway inhibitor-2 cDNA (accession number NM_006528). This sequence encoded for the entire C-terminal TFPI-2 protein (from amino acid residue 62) that included the last two Kunitz inhibitory domains (Fig. 1A). Interaction of TFPI-2 and ADAMTS1 in a Cell Culture Model—To confirm the ADAMTS1/TFPI-2 interaction, we performed co-immunoprecipitation studies in a mammalian cell-based system. 293T cells that constitutively overexpressed ADAMTS1 were transiently transfected with a TFPI-2-Myc chimera construct (scheme in Fig. 1B) or a control vector. Conditioned medium from these cells was independently subjected to immunoprecipitation with Myc and ADAMTS1 monoclonal antibodies (Fig. 2A). Both α and β TFPI-2 isoforms were pulled down with the Myc antibody; the γ TFPI-2 isoform, however, was not adequately resolved. In addition, p87-ADAMTS1 was observed to co-immunoprecipitate with the Myc antibody in the presence of TFPI-2, suggesting a specific interaction between these two proteins. To avoid antibody cross-reactivity, Western blot analyses were done with rabbit and guinea pig polyclonal antibodies to detect TFPI-2 and ADAMTS1 proteins, respectively. When the ADAMTS1 monoclonal antibody that recognizes all the forms of the protease was used in immunoprecipitation experiments, we observed the co-precipitation of both α and β TFPI-2 isoforms; again, the γ TFPI-2 isoform was not identified (Fig. 2A). Also, in this experiment, the antibodies used for the final detection were different from the ones used to pull down the complexes. Further validation of the interaction was achieved on 293T parental cells previously treated with phorbol esters (PMA). This stimulus is known to induce the expression of TFPI-2 (30Iochmann S. Reverdiau-Moalic P. Hube F. Bardos P. Gruel Y. Thromb. Res. 2002; 105: 217-223Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar), and here we demonstrate that it also induces ADAMTS1 expression. For these assays conditioned medium from 293T cells that were either treated or not treated with PMA for 48 h was harvested and immunoprecipitated with the ADAMTS1 monoclonal antibody. As expected, the analysis of these complexes revealed the presence of both ADAMTS1 and TFPI-2 only when the cells were stimulated with PMA (Fig. 2B). TFPI-2 Is a Substrate of ADAMTS1—TFPI-2 is a broad-spectrum protease inhibitor that targets both serine proteases and matrix metalloproteases. Thus, we first speculated that TFPI-2 could function as an endogenous inhibitor of ADAMTS1. To test this hypothesis, we evaluated the effect of TFPI-2 on aggrecan and syndecan-4 proteolytic assays, because these proteoglycans are known substrates for ADAMTS1 (9Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (205) Google Scholar). 3J. C. Rodriguez-Manzaneque, D. Carpizo, M. C. Plaza-Calonge, A. X. Torres-Collado, S. N.-M. Thai, M. Simons, A. Horowitz, and M. L. Iruela-Arispe, submitted for publication. However, the presence of TFPI-2 did not alter the proteolytic activity of ADAMTS1 on these substrates (data not shown). We then considered the potential effect of ADAMTS1 on TFPI-2. Cells overexpressing the inhibitor were transfected with either wild-type ADAMTS1 or the catalytically inactive form (zinc-binding site mutant E385A-ADAMTS1 (9Rodriguez-Manzaneque J.C. Westling J. Thai S.N. Luque A. Knauper V. Murphy G. Sandy J.D. Iruela-Arispe M.L. Biochem. Biophys. Res. Commun. 2002; 293: 501-508Crossref PubMed Scopus (205) Google Scholar)). As seen in Fig. 3A, last three lanes, the presence of ADAMTS1 resulted in increased levels of TFPI-2 in the conditioned media together with an apparent shift in the pattern of secreted isoforms. In contrast, this effect was not observed on cells transfected with the catalytically inactive ADAMTS1. To better understand this process, we used two types of arrows to indicate the different states of TFPI-2 isoforms; the black arrow indicates the primary TFPI-2 products, and the white arrow corresponds to the forms originated in the presence of ADAMTS1. Stars next to the bands also denote the new forms. We evaluated TFPI-2 levels in the presence of heparin, known to release ECM-bound and cell surface-anchored TFPI-2 (19Liu Y. Stack S.M. Lakka S.S. Khan A.J. Woodley D.T. Rao J.S. Rao C.N. Arch. Biochem. Biophys. 1999; 370: 112-118Crossref PubMed Scopus (33) Google Scholar). Although the overall quantities of soluble TFPI-2 were equivalent under these conditions, the mentioned changes in the pattern of TFPI-2-secreted isoforms persisted in the presence of ADAMTS1 (Fig. 3A). When the cell layer compartment was analyzed, the α-isoform of TFPI-2 was found to be predominant (Fig. 3A). A slight decrease of this form was observed in the presence of ADAMTS1, but no evidence of additional products appeared, which is indicative of the extracellular nature of this event. According to these data, we further explored the possibility that these changes in TFPI-2 are due to proteolysis by ADAMTS1. It has been previously reported that differences between TFPI-2 isoforms are due to post-translational glycosylation events (31Rao C.N. Reddy P. Liu Y. O'Toole E. Reeder D. Foster D.C. Kisiel W. Woodley D.T. Arch. Biochem. Biophys. 1996; 335: 82-92Crossref PubMed Scopus (88) Google Scholar). To facilitate our evaluation, the glycosylation inhibitor tunicamycin was included in this analysis. Tunicamycin treatment resulted in the appearance of two main bands of TFPI-2 (Fig. 3B); that is, the highly glycosylated α isoform that appears more abundantly in normal c" @default.
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• W2076304369 title "ADAMTS1 Interacts with, Cleaves, and Modifies the Extracellular Location of the Matrix Inhibitor Tissue Factor Pathway Inhibitor-2" @default.
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• W2076304369 doi "https://doi.org/10.1074/jbc.m513465200" @default.
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