Binding of ADAM28 to P-selectin Glycoprotein Ligand-1 Enhances P-selectin-mediated Leukocyte Adhesion to Endothelial Cells
2007; Elsevier BV; Volume: 282; Issue: 35 Linguagem: Inglês
10.1074/jbc.m702414200
ISSN1083-351X
AutoresMasayuki Shimoda, Gakuji Hashimoto, Satsuki Mochizuki, Eiji Ikeda, Norihiro Nagai, Susumu Ishida, Yasunori Okada,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions. ADAMs (a disintegrin and metalloproteinases) are a recently discovered gene family of multifunctional proteins with the disintegrin-like and metalloproteinase domains. To analyze the biological functions of ADAM28, we screened binding molecules to secreted-type ADAM28 (ADAM28s) by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 was determined by yeast two-hybrid assays, binding assays of the domain-specific recombinant ADAM28s species using PSGL-1 stable transfectants and leukocyte cell lines expressing native PSGL-1 (HL-60 cells and Jurkat cells), and co-immunolocalization and co-immunoprecipitation of the molecules in these cells. Incubation of HL-60 cells with recombinant ADAM28s enhanced the binding to P-selectin-coated wells and P-selectin-expressing endothelial cells. In addition, intravenous injection of ADAM28s-treated HL-60 cells increased their accumulation in the pulmonary microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. These data suggest a novel function that ADAM28s promotes PSGL-1/P-selectin-mediated leukocyte rolling adhesion to endothelial cells and subsequent infiltration into tissue spaces through interaction with PSGL-1 on leukocytes under inflammatory conditions. A disintegrin and metalloproteinases (ADAMs) 2The abbreviations used are:ADAMa disintegrin and metalloproteinaseEGFepidermal growth factorIGFBP-3insulin-like growth factor binding protein-3PSGL-1P-selectin glycoprotein ligand-1Disdisintegrin-likeCRcysteine-richSSsecreted-specificmAbmonoclonal antibodyAbantibodyrrecombinantPropropeptideMetmetalloproteinaseFITCfluorescein isothiocyanateBSAbovine serum albuminBCECF-AM2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) esterHUVECshuman umbilical vein endothelial cellsLPSlipopolysaccharideBALbronchoalveolar lavage. 2The abbreviations used are:ADAMa disintegrin and metalloproteinaseEGFepidermal growth factorIGFBP-3insulin-like growth factor binding protein-3PSGL-1P-selectin glycoprotein ligand-1Disdisintegrin-likeCRcysteine-richSSsecreted-specificmAbmonoclonal antibodyAbantibodyrrecombinantPropropeptideMetmetalloproteinaseFITCfluorescein isothiocyanateBSAbovine serum albuminBCECF-AM2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) esterHUVECshuman umbilical vein endothelial cellsLPSlipopolysaccharideBALbronchoalveolar lavage. are a recently discovered gene family of membrane-anchored and secreted proteins that have proteolytic and/or adhesive properties (1Primakoff P. Myles D.G. Trends Genet. 2000; 16: 83-87Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 2Blobel C.P. Nat. Rev. Mol. Cell Biol. 2005; 6: 32-43Crossref PubMed Scopus (902) Google Scholar). At present, more than 30 members have been identified in humans (3White J.M. Curr. Opin. Cell Biol. 2003; 15: 598-606Crossref PubMed Scopus (346) Google Scholar, 4Okada Y. Harris E.D. Budd R.C. Genovese M.C. Firestein G.S. Sargent J.S. Kelley's Textbook of Rheumatology. 7th Ed. Elsevier/Saunders, Philadelphia2005: 63-81Google Scholar). The precursor forms of ADAMs (pro-ADAMs) are composed of propeptide, metalloproteinase, disintegrin-like, cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and cytoplasmic domains (4Okada Y. Harris E.D. Budd R.C. Genovese M.C. Firestein G.S. Sargent J.S. Kelley's Textbook of Rheumatology. 7th Ed. Elsevier/Saunders, Philadelphia2005: 63-81Google Scholar, 5Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (438) Google Scholar). Roles of ADAMs include cell surface processing of membrane proteins such as tumor necrosis factor-α by ADAM17 (6Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2656) Google Scholar), heparin-binding epidermal growth factor by ADAM9 (7Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (472) Google Scholar), ADAM12 (8Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (638) Google Scholar), and ADAM17 (9Hinkle C.L. Sunnarborg S.W. Loiselle D. Parker C.E. Stevenson M. Russell W.E. Lee D.C. J. Biol. Chem. 2004; 279: 24179-24188Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and amyloid precursor protein by ADAM10 (10Allinson T.M. Parkin E.T. Turner A.J. Hooper N.M. J. Neurosci. Res. 2003; 74: 342-352Crossref PubMed Scopus (372) Google Scholar) and ADAM17 (11Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (974) Google Scholar). ADAMs are also reported to digest various proteins, including type IV collagen by ADAM10 (12Millichip M.I. Dallas D.J. Wu E. Dale S. McKie N. Biochem. Biophys. Res. Commun. 1998; 245: 594-598Crossref PubMed Scopus (109) Google Scholar) and insulin-like growth factor binding protein-3 (IGFBP-3) by ADAM12 (13Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (274) Google Scholar) and ADAM28 (14Mochizuki S. Shimoda M. Shiomi T. Fujii Y. Okada Y. Biochem. Biophys. Res. Commun. 2004; 315: 79-84Crossref PubMed Scopus (101) Google Scholar). On the other hand, accumulated lines of evidence have shown that the disintegrin-like domain of many ADAM members interacts with integrins (3White J.M. Curr. Opin. Cell Biol. 2003; 15: 598-606Crossref PubMed Scopus (346) Google Scholar, 15Reiss K. Ludwig A. Saftig P. Pharmacol. Ther. 2006; 111: 985-1006Crossref PubMed Scopus (100) Google Scholar), although a recent study on crystal structures suggested that the integrin-binding motif within the disintegrin-like domain is structurally inaccessible for protein binding (16Takeda S. Igarashi T. Mori H. Araki S. EMBO J. 2006; 25: 2388-2396Crossref PubMed Scopus (160) Google Scholar). The secreted form of ADAM9 has recently been reported to lead to invasion by binding to α6β4 and α2β1 integrins (17Mazzocca A. Coppari R. De Franco R. Cho J.Y. Libermann T.A. Pinzani M. Toker A. Cancer Res. 2005; 65: 4728-4738Crossref PubMed Scopus (151) Google Scholar). The cysteine-rich domain of ADAM12 is known to interact with syndecans on mesenchymal cells, leading to the β1 integrin-mediated cell spreading (18Thodeti C.K. Albrechtsen R. Grauslund M. Asmar M. Larsson C. Takada Y. Mercurio A.M. Couchman J.R. Wewer U.M. J. Biol. Chem. 2003; 278: 9576-9584Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The disintegrin-like and cysteine-rich domains of ADAM13 bind to laminin and fibronectin (19Gaultier A. Cousin H. Darribere T. Alfandari D. J. Biol. Chem. 2002; 277: 23336-23344Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, information about the binding molecules of ADAMs and their functional modulation by these interactions is still limited. a disintegrin and metalloproteinase epidermal growth factor insulin-like growth factor binding protein-3 P-selectin glycoprotein ligand-1 disintegrin-like cysteine-rich secreted-specific monoclonal antibody antibody recombinant propeptide metalloproteinase fluorescein isothiocyanate bovine serum albumin 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester human umbilical vein endothelial cells lipopolysaccharide bronchoalveolar lavage. a disintegrin and metalloproteinase epidermal growth factor insulin-like growth factor binding protein-3 P-selectin glycoprotein ligand-1 disintegrin-like cysteine-rich secreted-specific monoclonal antibody antibody recombinant propeptide metalloproteinase fluorescein isothiocyanate bovine serum albumin 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein tetrakis(acetoxymethyl) ester human umbilical vein endothelial cells lipopolysaccharide bronchoalveolar lavage. ADAM28 is expressed by human peripheral blood lymphocytes in two alternative forms, i.e. a prototype membrane-anchored form (ADAM28m) and a secreted form (ADAM28s) (20Roberts C.M. Tani P.H. Bridges L.C. Laszik Z. Bowditch R.D. J. Biol. Chem. 1999; 274: 29251-29259Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The disintegrin-like domain of ADAM28 is reported to interact with integrins α4β1, α4β7 and α9β1 on lymphocytes in an activation-dependent manner of the integrins (21Bridges L.C. Tani P.H. Hanson K.R. Roberts C.M. Judkins M.B. Bowditch R.D. J. Biol. Chem. 2002; 277: 3784-3792Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 22Bridges L.C. Sheppard D. Bowditch R.D. Biochem. J. 2005; 387: 101-108Crossref PubMed Scopus (55) Google Scholar). The metalloproteinase domain of ADAM28 has the zinc-binding catalytic-site consensus sequence, and ADAM28 cleaves myelin basic protein (23Howard L. Zheng Y. Horrocks M. Maciewicz R.A. Blobel C. FEBS Lett. 2001; 498: 82-86Crossref PubMed Scopus (63) Google Scholar), CD23 ectodomain (24Fourie A.M. Coles F. Moreno V. Karlsson L. J. Biol. Chem. 2003; 278: 30469-30477Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), and IGFBP-3 (14Mochizuki S. Shimoda M. Shiomi T. Fujii Y. Okada Y. Biochem. Biophys. Res. Commun. 2004; 315: 79-84Crossref PubMed Scopus (101) Google Scholar). In addition, we have reported that ADAM28 is overexpressed in non-small cell lung carcinomas with correlations to carcinoma cell proliferation and lymph node metastasis (25Ohtsuka T. Shiomi T. Shimoda M. Kodama T. Amour A. Murphy G. Ohuchi E. Kobayashi K. Okada Y. Int. J. Cancer. 2006; 118: 263-273Crossref PubMed Scopus (83) Google Scholar), and we have recently shown that ADAM28 plays a role in breast carcinoma cell proliferation through enhanced bioavailability of insulin-like growth factor-I by selective digestion of IGFBP-3 of the insulin-like growth factor-I·IGFBP-3 complex (26Mitsui Y. Mochizuki S. Kodama T. Shimoda M. Ohtsuka T. Shiomi T. Chijiiwa M. Ikeda T. Kitajima M. Okada Y. Cancer Res. 2006; 66: 9913-9920Crossref PubMed Scopus (109) Google Scholar). Thus, these data suggest the possibility that ADAM28 is involved in various cellular and tissue reactions such as cell-cell and cell-matrix interactions, cell motility, shedding of cell surface proteins, and cell proliferation. However, little is known about the biological functions of human ADAM28 and their molecular mechanisms under pathophysiological conditions. In this study, we screened ADAM28s-interacting proteins by the yeast two-hybrid system and identified P-selectin glycoprotein ligand-1 (PSGL-1). Our data provide the evidence that the binding between the disintegrin-like domain of ADAM28s and the extracellular portion of PSGL-1 enhances PSGL-1/P-selectin-mediated cell adhesion to endothelial cells in vitro and the interaction promotes accumulation of PSGL-1-expressing cells in the lung microcirculation and alveolar spaces in a mouse model of endotoxin-induced inflammation. Based on our results, we propose a novel pathway by which ADAM28s is involved in the promotion of leukocyte rolling adhesion to blood vessel endothelial cells and the subsequent migration into tissue spaces under inflammatory conditions. Yeast Two-hybrid System—MATCHMAKER Gal4 two-hybrid system 3 and the MATCHMAKER human lung cDNA library were purchased from Clontech. cDNA fragment encoding the disintegrin-like (Dis), cysteine-rich (CR), and secreted-specific (SS) domains corresponding to Cys410-Arg540 of ADAM28s was amplified by PCR using human lung cDNA library with the corresponding primers (Table 1). The product was cloned into the pGBKT7 vector, generating pGBKT7-Dis/CR/SS. The pGBKT7-Dis/CR/SS plasmid was co-introduced into Saccharomyces cerevisiae strain AH109 with the human lung cDNA library according to our previous methods (27Hashimoto G. Shimoda M. Okada Y. J. Biol. Chem. 2004; 279: 32483-32491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Yeast transformants were plated and selected on medium lacking leucine, tryptophan, histidine, and adenine. Robust colonies >2 mm in diameter were restreaked onto the same agar plates and allowed to grow for 1 week. This restreaking step was repeated twice more, and plasmids were isolated and introduced into Escherichia coli strain DH5α according to the manufacturer's instructions. Clones harboring target cDNA were isolated, and cDNA sequences were determined using a Mega-Base 1000 DNA sequencer (Amersham Biosciences).TABLE 1PCR primers to construct vectorsVectorOligonucleotide sequencepGBKT7-Dis/CR/SSForward, 5′-GGAATTCCATATGTGTGGGAACCAGTTGGTG-3′Reverse, 5′-CCGGAATTCTCATCTGAAATGATTTTCCTTCG-3′pGBKT7-DisForward, 5′-GGAATTCCATATGTGTGGGAACCAGTTGGTG-3′Reverse, 5′-CCGGAATTGAGGGAAGCCATTGACTTGG-3′pGBKT7-CRForward, 5′-GGAATTCCATATGTGCCATCACGGGAAGG-3′Reverse, 5′-CCGGAATTCTGGTCCCCACAGCTCTGTG-3′pGBKT7-SSForward, 5′-GGAATTCCATATGGGTAGGAGGACAAATCCTTTCC-3′Reverse, 5′-CCGGAATTCAGCAACCCTAAAACCAACCTC-3′pCMVTag4a-PSGL-1Forward, 5′-CCGGAATTCGTGCCATGCCTCTGCAAC-3′Reverse, 5′-ACGCGTCGACAGGGAGGAAGCTGTGCAGG-3′pCMVTag4a-Pro/MetForward, 5′-GGAATTCCCCAGCATGTTGCAAGGTCTC-3′Reverse, 5′-ACGCGTCGACAATTGGAGTGGATATGATATCTGTAGG-3′pCMVTag4a-Dis/CR/SS Signal peptideForward, 5′-GGAATTCCCCAGCATGTTGCAAGGTCTC-3′Reverse, 5′-ACGCAGATCTAGCACTTACTGCAACAGAGAG-3′Dis/CR/SS domainsForward, 5′-ACGCAGATCTATTTGTGGGAACCAGTTGG-3′Reverse, 5′-ACGCGTCGACTCTGAAATGATTTTCCTTCGC-3′ Open table in a new tab Yeast Two-hybrid Assay—cDNA fragments encoding the Dis, CR, or SS domain of ADAM28s were amplified by PCR using pGBKT7-Dis/CR/SS plasmid with the corresponding primers (Table 1). The products were cloned into the pGBKT7 vector, generating pGBKT7-Dis, pGBKT7-CR, and pGBKT7-SS (Fig. 1A). These plasmids were co-introduced into strain AH109 with pACT2 vector containing a cDNA fragment encoding full-length PSGL-1 (pACT2-PSGL-1) and assayed according to our previous methods (27Hashimoto G. Shimoda M. Okada Y. J. Biol. Chem. 2004; 279: 32483-32491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Establishment of Stable Transfectants Expressing PSGL-1—cDNA encoding human full-length PSGL-1 was prepared by PCR from the cDNAs derived from HL-60 cells (a human myeloid cell line) using the corresponding primers (Table 1), and cloned into pCMVTag4a (Stratagene, La Jolla, CA), generating pCMVTag4a-PSGL-1. Stable transfectants expressing PSGL-1 or mock transfectants were prepared by transfection of pCMVTag4a-PSGL-1 or pCMVTag4a vector alone to COS7 cells as described previously (28Shiomi T. Inoki I. Kataoka F. Ohtsuka T. Hashimoto G. Nemori R. Okada Y. Lab. Investig. 2005; 85: 1489-1506Crossref PubMed Scopus (60) Google Scholar), and 35 stable transfectants were established. The expression of PSGL-1 and ADAM28 in COS7 cells, mock transfectants, and PSGL-1 transfectants was examined by immunoblotting with anti-PSGL-1 monoclonal antibody (mAb) (KPL1 mAb; Santa Cruz Biotechnology, Santa Cruz, CA), anti-ADAM28 mAb (297-2F3) specific to the metalloproteinase domain of ADAM28 (25Ohtsuka T. Shiomi T. Shimoda M. Kodama T. Amour A. Murphy G. Ohuchi E. Kobayashi K. Okada Y. Int. J. Cancer. 2006; 118: 263-273Crossref PubMed Scopus (83) Google Scholar) or anti-β-actin antibody (Ab) (A5441; Sigma), and by flow cytometry with KPL1 mAb or nonimmune mouse IgG (Santa Cruz Biotechnology) as described previously (28Shiomi T. Inoki I. Kataoka F. Ohtsuka T. Hashimoto G. Nemori R. Okada Y. Lab. Investig. 2005; 85: 1489-1506Crossref PubMed Scopus (60) Google Scholar). Expression and Purification of Recombinant ADAM28s Species—pCMVTag4a-pro-ADAM28s containing the full-length pro-ADAM28s cDNA with its signal peptide was previously prepared (14Mochizuki S. Shimoda M. Shiomi T. Fujii Y. Okada Y. Biochem. Biophys. Res. Commun. 2004; 315: 79-84Crossref PubMed Scopus (101) Google Scholar). pCMVTag4a-Pro/Met having a cDNA fragment encoding the signal peptide, propeptide (Pro), and metalloproteinase (Met) domains was prepared by PCR from pCMVTag4a-pro-ADAM28s using the corresponding primers (Table 1). cDNA fragments encoding the signal peptide and the Dis, CR, and SS domains were amplified by PCR using the pCMVTag4a-pro-ADAM28s plasmid and the corresponding primers (Table 1). The fragments were cloned into EcoRI/SalI-digested pCMV-Tag4a vector to fuse the signal peptide to the amino terminus of the Dis/CR/SS domains, generating pCMVTag4a-Dis/CR/SS. The FLAG-tagged cDNA fragments encoding ADAM28s species were subcloned into pFASTBac1 vector (Invitrogen), generating pFASTBac1-pro-ADAM28s, pFASTBac1-Pro/Met, and pFASTBac1-Dis/CR/SS. The vectors were infected to insect Sf9 cells, and the recombinant full-length pro-ADAM28s (rpro-ADAM28s) and its deletion mutants consisting of the Dis, CR, and SS domains (rDis/CR/SS) or the Pro and Met domains (rPro/Met) were purified by anti-FLAG M2-agarose affinity gels (Sigma) according to our previous methods (14Mochizuki S. Shimoda M. Shiomi T. Fujii Y. Okada Y. Biochem. Biophys. Res. Commun. 2004; 315: 79-84Crossref PubMed Scopus (101) Google Scholar). Binding Assay of rADAM28s Species to PSGL-1-expressing Cells—Parental COS7 cells, mock transfectants, and stable transfectants expressing PSGL-1 were incubated with 125I-labeled rpro-ADAM28s, rDis/CR/SS, or rPro/Met (1 nm each), and the percentage of bound activity to added activity was calculated by counting the radioactivity using a γ-counter (29Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K. Ikeda E. Takata S. Kobayashi K. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (323) Google Scholar). A similar binding assay was performed with HL-60 cells and Jurkat cells (a T-lymphoma cell line). The competitive inhibition study was performed by incubation of stable transfectants, HL-60 cells, and Jurkat cells with a 10-fold excess amount of nonlabeled rADAM28s species prior to the binding assay. The binding assay was also carried out in the presence of 1 μm KB-R7785, an ADAM inhibitor (a gift by Dr. Koichiro Yoshino, Carnabioscience, Kobe, Japan) (8Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (638) Google Scholar, 26Mitsui Y. Mochizuki S. Kodama T. Shimoda M. Ohtsuka T. Shiomi T. Chijiiwa M. Ikeda T. Kitajima M. Okada Y. Cancer Res. 2006; 66: 9913-9920Crossref PubMed Scopus (109) Google Scholar). For the inhibition studies, these cells were incubated with anti-PSGL-1 mAb (KPL1 mAb or PL1 mAb; Santa Cruz Biotechnology), anti-PSGL-1 polyclonal Ab (H-300 Ab; Santa Cruz Biotechnology), or non-immune IgG prior to the binding assays. Laser Scanning Confocal Microscopy—Cell suspensions of HL-60 and Jurkat cells were incubated with rpro-ADAM28s in phosphate-buffered saline containing 2% fetal bovine serum (JRH Bioscience, Lenexa, KS) or buffer alone. After incubation with anti-FLAG M2 mAb (Sigma) or nonimmune mouse IgG, they were plated on glass slides, fixed with methanol/acetone/formaldehyde (27Hashimoto G. Shimoda M. Okada Y. J. Biol. Chem. 2004; 279: 32483-32491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 28Shiomi T. Inoki I. Kataoka F. Ohtsuka T. Hashimoto G. Nemori R. Okada Y. Lab. Investig. 2005; 85: 1489-1506Crossref PubMed Scopus (60) Google Scholar), and incubated with goat anti-PSGL-1 polyclonal Ab (C-19 Ab; Santa Cruz Biotechnology) or nonimmune goat IgG (R & D System, Minneapolis, MN). Following incubation with fluorescein isothiocyanate (FITC)- or rhodamine-conjugated secondary Ab (Dako Corp., Glostrup, Denmark), all preparations were viewed under an Olympus laser scanning confocal microscope Fluoview FV300 (Olympus, Tokyo, Japan) (27Hashimoto G. Shimoda M. Okada Y. J. Biol. Chem. 2004; 279: 32483-32491Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Immunoprecipitation of rpro-ADAM28s·PSGL-1 Complex—HL-60 and Jurkat cells were incubated with 125I-labeled rpro-ADAM28s, and supernatants of the cell lysates were subjected to immunoprecipitation with KPL1 mAb, anti-FLAG M2 mAb, or nonimmune mouse IgG according to our previous methods (28Shiomi T. Inoki I. Kataoka F. Ohtsuka T. Hashimoto G. Nemori R. Okada Y. Lab. Investig. 2005; 85: 1489-1506Crossref PubMed Scopus (60) Google Scholar). The immunoprecipitates were immunoblotted with KPL1 mAb as described above. 125I-Labeled rpro-ADAM28s on the same membranes was detected by an imaging plate and the BAS-2000 system (Fuji Photo Film Co., Tokyo, Japan). Adhesion Assay of HL-60 Cells to Immobilized P-selectin—Prior to the adhesion assay, the glycosylation patterns of PSGL-1 in HL-60 and Jurkat cells were examined by flow cytometry using CSLEX-1 Ab (BD Biosciences) and NCC-ST-439 Ab (Nippon Kayaku Co., Tokyo, Japan), which recognize sialyl-Lewis X and both nonsulfated and 6-sulfated sialyl-Lewis X on core 2-type O-glycans, respectively. Microtiter plates (NalgeNunc, Rochester, NY) were coated with human P-selectin (R & D Systems) or bovine serum albumin fraction V (BSA; Sigma) in phosphate-buffered saline containing 1 mm CaCl2 and 1 mm MgCl2. Suspensions of HL-60 cells, which were labeled with 2′,7′-bis (2-carboxyethyl)-5 (6)-carboxyfluorescein tetrakis (acetoxymethyl) ester (BCECF-AM; Sigma) (30Hirose M. Kawashima H. Miyasaka M. Int. Immunol. 1998; 10: 639-649Crossref PubMed Scopus (12) Google Scholar), were incubated with rpro-ADAM28s, rDis/CR/SS, or rPro/Met and allowed to adhere to the microtiter plates for 10 min at 4 °C. The adherent cells were observed by an inverted microscope, and a percentage of adherent cells to added cells was calculated by quantitating the fluorescence intensity (Dainippon Sumitomo Pharma Co., Osaka, Japan). For inhibition studies, the cells were incubated with rpro-ADAM28s in the presence of H-300 Ab, KPL1 mAb, or nonimmune IgG prior to the adhesion assay. Similarly, the adhesion assay was performed with active rADAM28s (14Mochizuki S. Shimoda M. Shiomi T. Fujii Y. Okada Y. Biochem. Biophys. Res. Commun. 2004; 315: 79-84Crossref PubMed Scopus (101) Google Scholar). Shedding of PSGL-1 from HL-60 cells after treatment with rpro-ADAM28s or active rADAM28s was analyzed by flow cytometry using KPL1 mAb or nonimmune mouse IgG. Adhesion Assay of HL-60 Cells to Thrombin-stimulated Human Umbilical Vein Endothelial Cells (HUVECs)—Primary HUVECs were prepared (31Jaffe E.A. Nachman R.L. Becker C.G. Minick C.R. J. Clin. Investig. 1973; 52: 2745-2756Crossref PubMed Scopus (5968) Google Scholar) and characterized by the expression of factor VIII-related antigens and CD31 by immunohistochemistry (29Inoki I. Shiomi T. Hashimoto G. Enomoto H. Nakamura H. Makino K. Ikeda E. Takata S. Kobayashi K. Okada Y. FASEB J. 2002; 16: 219-221Crossref PubMed Scopus (323) Google Scholar, 32Villard E. Alonso A. Agrapart M. Challah M. Soubrier F. J. Biol. Chem. 1998; 273: 25191-25197Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). HUVECs in 24-well plates were stimulated without or with 1 unit/ml thrombin (Sigma) for 5 min and fixed with 1% paraformaldehyde (33Kameda H. Morita I. Handa M. Kaburaki J. Yoshida T. Mimori T. Murota S. Ikeda Y. Br. J. Haematol. 1997; 97: 348-355Crossref PubMed Scopus (45) Google Scholar). Suspensions of BCECF-AM-labeled HL-60 cells were reacted with rpro-ADAM28s, rDis/CR/SS, rPro/Met, active rADAM28s, or buffer alone, distributed over the plates of HUVECs, and incubated for 10 min at room temperature with shaking at 60 rpm with an orbital shaker (Taitec, Saitama, Japan) (33Kameda H. Morita I. Handa M. Kaburaki J. Yoshida T. Mimori T. Murota S. Ikeda Y. Br. J. Haematol. 1997; 97: 348-355Crossref PubMed Scopus (45) Google Scholar). Cells attached to HUVECs were observed by an inverted microscope, and the fluorescence intensity of the cell lysates was measured. Inhibition studies using anti-PSGL-1 Abs (H-300 Ab and KPL1 mAb) or nonimmune IgG were also performed. Adhesion and Transendothelial Migration Study of HL-60 Cells in a Mouse Model—Systemic inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS; Sigma) or phosphate-buffered saline in adult C57BL/6 mice (9-10 weeks; SLC, Shizuoka, Japan) 18 h prior to the experiments (34Friederichs J. Zeller Y. Hafezi-Moghadam A. Grone H.J. Ley K. Altevogt P. Cancer Res. 2000; 60: 6714-6722PubMed Google Scholar, 35Nagai N. Oike Y. Noda K. Urano T. Kubota Y. Ozawa Y. Shinoda H. Koto T. Shinoda K. Inoue M. Tsubota K. Yamashiro K. Suda T. Ishida S. Investig. Ophthalmol. Vis. Sci. 2005; 46: 2925-2931Crossref PubMed Scopus (75) Google Scholar). BCECF-AM-labeled HL-60 cells in RPMI 1640 medium (Sigma) containing 25 mm HEPES were incubated with rpro-ADAM28s, rDis/CR/SS, rPro/Met, or buffer alone and then injected into mouse tail veins. After 12 h, the lungs were resected under deep anesthesia. Frozen sections were examined by a fluorescence microscope, and the number of HL-60 cells per1mm2 of the lung tissues from six mice in each group was determined by counting fluorescent cells in at least 10 distinct sections per animal. For the inhibition study, mice were treated with neutralizing anti-P-selection mAb (RB40.34 mAb; BD Biosciences) 2 h before the cell injection (36Kunkel E.J. Jung U. Ley K. Am. J. Physiol. 1997; 272: H1391-H1400Crossref PubMed Google Scholar). To localize the endothelial cells, frozen sections fixed with 4% paraformaldehyde were subjected to immunofluorescent staining using rat anti-CD31 mAb (MEC13.3; BD Biosciences) and rhodamine-conjugated secondary Ab (Immunotech). Bronchoalveolar lavage (BAL) fluids were prepared (37Reutershan J. Basit A. Galkina E.V. Ley K. Am. J. Physiol. 2005; 289: L807-L815Crossref PubMed Scopus (251) Google Scholar), and the fluorescence intensity of the cell pellets was measured by plate reader. Care of the animals was in accordance with the Guidelines for the Care and Use of Laboratory Animals of Keio University School of Medicine, and our experiments have been approved by the University Animal Welfare Committee. Statistical Analysis—Results between the two independent groups were determined by the Student's t test. Comparisons among more than three groups were determined by the Bonferroni/Dunn test. Statistical analyses were carried out using StatView statistical software (SAS Institute Inc. Cary, NC) on a personal computer. p values less than 0.05 were considered significant. Screening of Proteins That Interact with ADAM28s and Determination of Its Interacting Domain—To seek binding proteins to ADAM28s, we screened 1.0 × 107 clones of human lung cDNA library by the yeast two-hybrid system using a cDNA fragment encoding the Dis, CR, and SS domains of ADAM28s as bait (Fig. 1A), and isolated 215 positive clones. Among the clones, five were identified as human PSGL-1; they encoded the decamer repeats, transmembrane and cytoplasmic domains of PSGL-1. Then we further performed yeast two-hybrid assays to determine the ADAM28s domain involved in the binding with PSGL-1. As shown in Fig. 1, A and B, plasmid DNAs encoding different domains of ADAM28s (pGBKT7-Dis/CR/SS, pGBKT7-Dis, pGBKT7-CR, and pGBKT7-SS) were co-introduced with that encoding full-length PSGL-1 (pACT2-PSGL-1) into S. cerevisiae strain AH109. The yeast transformants of pGBKT7-Dis/CR/SS or pGBKT7-Dis with pACT2-PSGL-1 and positive control transformants expressing p53 and SV40 showed definite growth on high stringency plates, whereas only negligible background growth was observed with those of pGBKT7-CR, pGBKT7-SS, or pGBKT7 vector alone (negative control) with pACT2-PSGL-1 (Fig. 1B). When the α-galactosidase activity of each transformants was measured, the transformants of pGBKT7-Dis/CR/SS or pGBKT7-Dis with pACT2-PSGL-1 showed higher activity as compared with other transformants (Fig. 1C). These data strongly suggest that the Dis domain of ADAM28s interacts with PSGL-1. Binding of Recombinant ADAM28s Species to PSGL-1-expressing Cells—The expression levels of PSGL-1 in th
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