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• W2094395747 abstract "Early metastatic growth occurs at sites of vascular arrest of blood-borne cancer cells and is entirely intravascular. Here we show that lung colonization by B16-F10 cells is licensed by β4 integrin adhesion to the mouse lung endothelial Ca2+-activated chloride channel protein mCLCA1. In a manner independent of Met, β4integrin-mCLCA1-ligation leads to complexing with and activation of focal adhesion kinase (FAK) and downstream signaling to extracellular signal-regulated kinase (ERK). FAK/ERK signaling is Src-dependent and is interrupted by adhesion blocking antibodies and by dominant-negative (dn)-FAK mutants. Levels of ERK activation in B16-F10 cells transfected with wild-type or mutant FAK are closely associated with rates of proliferation and bromodeoxyuridine (BrdUrd) incorporation of tumor cells grown in mCLCA1-coated dishes, the ability to form tumor cell colonies on CLCA-expressing endothelial cell monolayers, and the extent of pulmonary metastatic growth. Parallel with the transfection rates, B16-F10 cells transfected with dn-FAK mutants and injected intravenously into syngeneic mice generate approximately half the number and size of lung colonies that vector-transfected B16-F10 cells produce. For the first time, β4 integrin ligation to its novel CLCA-adhesion partner is shown to be associated with FAK complexing, activation, and signaling to promote early, intravascular, metastatic growth. Early metastatic growth occurs at sites of vascular arrest of blood-borne cancer cells and is entirely intravascular. Here we show that lung colonization by B16-F10 cells is licensed by β4 integrin adhesion to the mouse lung endothelial Ca2+-activated chloride channel protein mCLCA1. In a manner independent of Met, β4integrin-mCLCA1-ligation leads to complexing with and activation of focal adhesion kinase (FAK) and downstream signaling to extracellular signal-regulated kinase (ERK). FAK/ERK signaling is Src-dependent and is interrupted by adhesion blocking antibodies and by dominant-negative (dn)-FAK mutants. Levels of ERK activation in B16-F10 cells transfected with wild-type or mutant FAK are closely associated with rates of proliferation and bromodeoxyuridine (BrdUrd) incorporation of tumor cells grown in mCLCA1-coated dishes, the ability to form tumor cell colonies on CLCA-expressing endothelial cell monolayers, and the extent of pulmonary metastatic growth. Parallel with the transfection rates, B16-F10 cells transfected with dn-FAK mutants and injected intravenously into syngeneic mice generate approximately half the number and size of lung colonies that vector-transfected B16-F10 cells produce. For the first time, β4 integrin ligation to its novel CLCA-adhesion partner is shown to be associated with FAK complexing, activation, and signaling to promote early, intravascular, metastatic growth. human embryonic kidney reverse transcription focal adhesion kinase proline-rich tyrosine kinase-2 phosphatidylinositol 3-kinase extracellular signal-regulated kinase bovine aortic endothelial cells dominant-negative monoclonal antibody polyclonal antibody hemagglutinin phosphotyrosine Dulbecco's modified Eagle's medium fetal bovine serum wild-type green fluorescent protein poly-l-lysine bovine serum albumin octylglucoside horseradish peroxidase phosphate-buffered saline myelin basic protein immunoprecipitation Western blotting FRAK-related non-kinase Englbreth-Hom-Swarm phospho-MBP Hematogenous metastases originate from tumor cells arrested in the vasculature of select target organs. This arrest is tumor- and tissue-specific and is mediated at least in part by distinct tumor cell/endothelial cell adhesion ligand/receptor pairs (reviewed in Refs.1Pauli B.U. Lin H. Bertino J.R. Encyclopedia of Cancer. Academic Press, San Diego, CA1997: 464-476Google Scholar and 2Orr F.W. Wang H.H. Lafrenie R.M. Scherbarth S. Nance D.M. J. Pathol. 2000; 190: 310-329Crossref PubMed Scopus (253) Google Scholar). Studies in our laboratory have shown that lung metastatic human breast cancer cells colonized the lungs following adhesion to hCLCA2, a Ca2+-sensitive chloride channel protein that is expressed on the endothelial cell luminal surface of human pulmonary arteries, arterioles, and interlobular venules (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Similar to the unique adhesion functions of other channel proteins (reviewed in Ref. 4Pauli B.U. Cheng H.C. Abdel-Ghany M. Fuller C.M. Ca2+-activated Cl-Channels. Current Topics in Membranes. Academic Press, San Diego, CA2002Google Scholar), CLCA proteins mediate adhesion via the β4integrin tumor cell ligand, which for the first time has been associated with a cell-cell adhesion function (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 4Pauli B.U. Cheng H.C. Abdel-Ghany M. Fuller C.M. Ca2+-activated Cl-Channels. Current Topics in Membranes. Academic Press, San Diego, CA2002Google Scholar). This novel adhesion interaction between members of CLCA family of proteins (e.g. bCLCA2 (Lu-ECAM-1), hCLCA2) and the β4integrin has been scrutinized by a variety of stringently controlled biochemical and functional assay procedures (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 6Zhu D. Cheng C.F. Pauli B.U. J. Clin. Invest. 1992; 89: 1718-1724Crossref PubMed Scopus (51) Google Scholar). These assays included (i) the co-immunoprecipitation of the β4-hCLCA2 complex from extracts of lung metastatic MDA-MB-231 breast cancer cells bound to monolayers of hCLCA2-transfected human embryonic kidney (HEK)1 293 cells (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar); (ii) the selective binding of immunopurified, recombinant hCLCA2 to membrane-immobilized, reconstituted β4 integrin in Far Westerns (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar); (iii) the increased expression of the β4integrin in breast cancer cell lines selected in vivo for enhanced lung colonization and the concomitant increased adhesion of the selected cells to hCLCA2 (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar); (iv) the loss of hCLCA2 adhesion of breast cancer cells subjected to selective cleavage of the β4 integrin with the metalloproteinase matrilysin (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 7Von Bredow D.C. Nagle R.B. Bowden G.T. Cress A.E. Exp. Cell Res. 1997; 236: 341-345Crossref PubMed Scopus (107) Google Scholar) or tumor cells transfected with mutant β4 integrin (tail-less) (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar); (v) the inhibition of pulmonary metastases by β4-hCLCA2 adhesion-blocking antibodies directed against either of the interacting adhesion molecules (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 6Zhu D. Cheng C.F. Pauli B.U. J. Clin. Invest. 1992; 89: 1718-1724Crossref PubMed Scopus (51) Google Scholar); and (vi) the increased lung metastatic performance of tumor cells overexpressing the β4 integrin (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). These data are in agreement with previous reports showing that overexpression of the β4integrin is associated with an aggressive/metastatic cancer phenotype in several malignancies (reviewed in Ref. 8Rabinovitz I. Mercurio A.M. Biochem. Cell Biol. 1996; 74: 811-821Crossref PubMed Scopus (103) Google Scholar) and, more recently, with the finding that selection for enhanced lung colonization concurs with prominent overexpression of the β4 integrin gene in a murine metastasis model analyzed by cDNA microarray (9Khanna C. Khan J. Nguyen P. Prehn J. Caylor J. Yeung C. Trepel J. Meltzer P. Helman L. Cancer Res. 2001; 61: 3750-3759PubMed Google Scholar). Here, B16-F10 melanoma cells, characterized by strong surface expression of the β4 integrin (10Falcioni R. Kennel S.J. Giacomini P. Zupi G. Sacchi A. Cancer Res. 1986; 46: 5772-5778PubMed Google Scholar, 11Cimino L. Perrotti D. D'Agostino G. Falcioni R. Sacchi A. Cancer Detect. Prev. 1997; 21: 158-166PubMed Google Scholar) and consistently high lung colonization potential (12Fidler I.J. Nat. New Biol. 1973; 242: 148-149Crossref PubMed Scopus (1277) Google Scholar), were employed to explore whether the β4 integrin interacts with a murine CLCA family member to promote metastasis of mouse lungs. Although the existence of a murine counterpart of hCLCA2 has been suspected by the positive immunohistochemical staining of endothelia of mouse pulmonary blood vessels with anti-bCLCA2 mAb6D3 and by the anti-metastatic effect of mAb6D3 (5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 6Zhu D. Cheng C.F. Pauli B.U. J. Clin. Invest. 1992; 89: 1718-1724Crossref PubMed Scopus (51) Google Scholar, 13Zhu D. Pauli B.U. Int. J. Cancer. 1993; 53: 628-633Crossref PubMed Scopus (20) Google Scholar, 14Gerritsen M.E. Shen C.-P. Mchugh M.C. Atkinson W.J. Kiely J.-M. Milstone D.S. Luscinskas F.W. Gimbrone M.A., Jr. Microcirculation. 1995; 2: 151-163Crossref PubMed Scopus (62) Google Scholar), cloning of this molecule was not achieved due to unavailability of a pure pulmonary endothelial cell source. We report now the isolation and cloning of this molecule, which establish identity with the previously cloned mCLCA1 (15Gandhi R. Elble R.C. Gruber A.D. Schreur K.D., Ji, H.L. Fuller C.M. Pauli B.U. J. Biol. Chem. 1998; 273: 32096-32101Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), illustrate the mCLCA1 expression pattern in the lung vasculature both by RT-PCR and by immunohistochemistry, and characterize the specific binding interaction between mCLCA1 and murine β4 integrin. Intrigued by studies in a novel pulmonary metastasis model that allowed the in situ tracking of blood-borne cancer cells in perfused rodent lungs and the finding that lung metastases arose exclusively from endothelial cell-bound tumor cells by intravascular growth (16Al Mehdi A.B. Tozawa K. Fisher A.B. Shientag L. Lee A. Muschel R.J. Nat. Med. 2000; 6: 100-102Crossref PubMed Scopus (581) Google Scholar), we then explored whether the β4-mCLCA1 adhesion by activating distinct, growth-promoting signaling pathways could account for the observed intravascular tumor cell proliferation. Our effort was focused on three signaling targets that may operate immediately downstream of the establishment of focal adhesions and promote cell growth. These targets were focal adhesion kinase (FAK), proline-rich tyrosine kinase-2 (Pyk2), and phosphatidylinositol 3-kinase (PI3K) (reviewed in Refs. 17Schlaepfer D.D. Hauck C.R. Sieg D.J. Mol. Biol. 1999; 71: 4356-4378Google Scholar, 18Zhao J.-H. Guan J.-L. Progr. Mol. Subcell. Biol. 2000; 25: 37-55Crossref PubMed Scopus (23) Google Scholar, 19Mercurio A.M. Rabinovitz I. Shaw L.M. Curr. Opin. Cell Biol. 2001; 13: 541-545Crossref PubMed Scopus (243) Google Scholar). We found that β4 integrin ligation to mCLCA1 selectively caused complexing with and activation of FAK that did not require participation of the Met oncogene (20Trusolino L. Bertotti A. Comoglio P.M. Cell. 2001; 107: 643-654Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Downstream of FAK the extracellular signal-regulated kinase (ERK) was activated to promote tumor cell proliferation on surfaces coated with recombinant mCLCA1 and on bovine aortic endothelial cells (BAEC) that constitutively express bCLCA2 protein (5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar). FAK/ERK signaling was abrogated by β4-mCLCA1 adhesion-blocking antibodies and by transfection of B16-F10 with dominant-negative (dn)-FAK mutants. These dn-FAK mutants also suppressed the metastatic growth of B16-F10 cells by down-regulating intravascular tumor cell proliferation, showing for the first time that FAK signaling initiated by tumor cell β4 integrin ligation to its novel endothelial cell mCLCA1adhesion partner is critical during the initial steps of metastasis formation. Antibodies against the β4 integrin ectodomain were rat mAb346-11A (BD PharMingen, San Diego, CA) and mouse mAb3E1 (Dr. E. Engvall, The Burnham Institute, La Jolla, CA), and against the β4cytoplasmic domain rabbit pAb1922 (Chemicon, Temecula, CA) was used. Mouse mAb9E10 was against the Myc protein tag (Calbiochem, San Diego, CA), rabbit pAb (α-FAK) against chicken FAK (Dr. J. L. Guan, Cornell University, Ithaca, NY), mouse mAb clone BU-33 against BrdUrd (Sigma Chemical Co., St. Louis, MO), rabbit pAb against the glutamate receptor subunit GluR3 (21Carson N.G. Gahring L.C. Twyman R.E. Rogers S.W. J. Biol. Chem. 1997; 272: 11295-11301Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) (Dr. L. Nowak, Cornell University, Ithaca, NY), mouse mAb against the HA(F-7) tag, rabbit pAbH-102 against Pyk2, rabbit pAb H-239 against PI3K (p110), rabbit pAb N-16 against Src, mouse mAbB2 against Met, and mouse mAbPY20 against phosphotyrosine (pY) (all from Santa Cruz Biotechnology, Santa Cruz, CA), all cross-reacting with the respective mouse counterpart proteins. Agarose-conjugated anti-ERK rabbit pAbK-23 was from Santa Cruz Biotechnology. Anti-bCLCA2 (Lu-ECAM-1) mAb6D3 was produced in BALB/c mice (22Zhu D. Pauli B.U. J. Histochem. Cytochem. 1991; 39: 1137-1142Crossref PubMed Scopus (26) Google Scholar) and selected for blocking the adhesion of B16-F10 melanoma cells to bCLCA2-expressing BAEC (5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 6Zhu D. Cheng C.F. Pauli B.U. J. Clin. Invest. 1992; 89: 1718-1724Crossref PubMed Scopus (51) Google Scholar). The antibody cross-reacts with mCLCA1 (5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 13Zhu D. Pauli B.U. Int. J. Cancer. 1993; 53: 628-633Crossref PubMed Scopus (20) Google Scholar, 14Gerritsen M.E. Shen C.-P. Mchugh M.C. Atkinson W.J. Kiely J.-M. Milstone D.S. Luscinskas F.W. Gimbrone M.A., Jr. Microcirculation. 1995; 2: 151-163Crossref PubMed Scopus (62) Google Scholar). Rat pAb4 (rat4) was against the gel-excised 90-kDa protein of bCLCA2 (23Elble R. Widom J. Gruber A.D. Abdel-Ghany M. Levine R. Goodwin A. Cheng H.-C. Pauli B.U. J. Biol. Chem. 1997; 272: 27853-27861Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Rat plasma fibronectin was from Invitrogen (Grand Island, NY). All other reagents were from Sigma Chemical Co. B16-F10 melanoma cells were obtained from Dr. I. J. Fidler (MD Anderson Cancer Center, Houston, TX). BAEC were isolated from thoracic aortas of 18-month-old steers and used as monolayers grown on plastic or lung matrix extracts (24Pauli B.U. Lee C.L. Lab. Invest. 1988; 58: 379-387PubMed Google Scholar). HEK293 cells were from ATCC (Manassas, VA). All cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Expression constructs encoding HA-tagged wild-type (wt) chicken FAK (pKH3-FAK-HA) or the dn-FAK mutants FRNK (FAK-related non-kinase) (pKH3-FRNK-HA) and FAKY397F (pKH3-FAKY397F-HA) were obtained from Dr. J. L. Guan (25Zhao J.-H. Reiske H. Guan J.-L. J. Cell Biol. 1998; 143: 1997-2008Crossref PubMed Scopus (302) Google Scholar). B16-F10 cells were transiently transfected with pKH3-FAK-HA, pKH3-FRNK-HA, pKH3-FAKY397F-HA, or vector alone, using LipofectAMINE Plus as described by the manufacturer (Invitrogen). Transfection rates assessed by GFP co-transfection were 40–50%. Cells were used in the various assays 48–120 h after transfection unless otherwise stated. RNA was prepared from frozen, powdered mouse lungs and from cultures of mouse pulmonary endothelial cells (Dr. M. E. Gerritsen, Genentech, South San Francisco, CA) and mouse aortic endothelial cells (Dr. B. Nilius, CU Leuven, Leuven, Belgium) by extraction with TRIzol (Invitrogen). B16-F10 and HEK293 cells served as negative controls. 1 μg of RNA was reverse-transcribed (Superscript, Life Technologies) using random hexamers. cDNA was subjected to PCR (93 °C, 30 min; 55 °C, 30 min; 72 °C, 30 min; 35 cycles) with degenerate primers based on bCLCA2 (Lu-ECAM-1) amino acids 36–45 (5′-ATTGCAATTAACCCCAGTGTGCCAGANGA-3′) and 165–174 (5′-GCRTAYTCRTCRAANAYNCCCCA-3′) (26Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 40510-40517Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). The 414-bp PCR products were inserted into pGEM-T (Promega) and sequenced. Surface-biotinylated B16-F10 melanoma cells were bound to monolayers of either HEK293 cells transfected with mCLCA1 or BAEC constitutively expressing bCLCA2 for 30 min at 37 °C in DMEM containing 1% BSA (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Cells were lysed in TBS-lysis buffer (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm phenylmethylsulfonyl fluoride, 0.01% aprotinin, 1 mm benzamidine, and 1% octylglucoside (OG); 1 h at 4 °C), and lysates were centrifuged at 15,000 rpm (20 min at 4 °C) to remove insoluble materials. Precleared supernatants were mixed with anti-Myc mAb9E10 and incubated for 4 h at 4 °C. Protein G-Sepharose beads were then added to the reaction mixture and incubated overnight at 4 °C. Immune complexes were washed extensively with cold TBS lysis buffer (0.5% OG) and analyzed by 8% SDS-PAGE and Western blotting, using streptavidin-HRP and anti-β4 pAb1922. Cultures of B16-F10 and mCLCA1-transfected HEK293 cells served as controls. Pull-down assays were performed essentially as described by Puzon-McLaughlin and Takada (27Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). In brief, β4 integrin immunopurified from B16-F10 or HEK293 cells co-transfected with the α6 and β4 integrin subunits was immobilized on Protein G-Sepharose beads conjugated with anti-β4 pAb1922 (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Beads with bound β4integrin were washed extensively with lysis buffer containing 1 mm CaCl2, 2 mm MgCl2, and 0.5%, instead of 1% OG (washing buffer), then incubated overnight at 4 °C with mCLCA1 immunopurified from HEK293 cells transfected with mCLCA1-Myc or lysates from surface-biotinylated BAEC, which constitutively express bCLCA2 (both CLCA preparations in TBS lysis buffer containing 1 mm CaCl2 and 2 mm MgCl2 at a final detergent concentration of 0.5% OG) (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 27Puzon-McLaughlin W. Takada Y. J. Biol. Chem. 1996; 271: 20438-20443Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Conversely, beads conjugated with anti-Myc mAb9E10 and bound mCLCA1-Myc were used to pull-down the β4integrin from lysates of HEK293 cells co-transfected with the α6 and β4 integrin subunits or surface-biotinylated B16-F10 cells (both cell lysates prepared in the same 0.5% OG-containing buffer as above). For detection of bound protein, beads were washed extensively with washing buffer and boiled in SDS sample buffer, and bound material was detected by SDS-PAGE and Western blotting. The glutamate receptor subunit GluR3, a multimembrane-spanning channel protein with similar hydrophobicity characteristics as CLCA proteins was used as negative control (21Carson N.G. Gahring L.C. Twyman R.E. Rogers S.W. J. Biol. Chem. 1997; 272: 11295-11301Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Wild-type (wt) mCLCA1 protein was purified by mAb6D3-immunaffinity chromatography from lung extracts, following surface biotinylation of pulmonary endothelia by in situ perfusion of mice via the right heart ventricle (28Cheng H.-C. Abdel-Ghany M. Zhang S. Pauli B.U. Clin. Exp. Metastasis. 1999; 17: 609-615Crossref PubMed Scopus (31) Google Scholar). Recombinant Myc-tagged mCLCA1 was prepared from extracts of HEK293 transfected with mCLCA1-Myc, using mAb9E10 (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). β4-mCLCA1-mediated signaling protocols were carried out with B16-F10 cells that had been serum-starved for 24 h in DMEM. Serum-starved tumor cells were removed from the growth surface with 5 mm EDTA in PBS, washed thrice in DMEM containing 1% BSA, and immediately seeded into 35-mm dishes (1 × 106cells/dish) previously coated with either mCLCA1 (∼3 μg/ml) or poly-l-lysine (PLL, 1 mg/ml) and blocked with 2% BSA in PBS. Dishes coated with EHS laminin and placental laminin (both 20 μg/ml) served as control substrates for the β4 integrin (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). B16-F10 cells were incubated for various periods of time, and bound tumor cells were lysed in modified TBS lysis buffer containing 1 mm EDTA, 1 mm EGTA, 10 mm NaF, 0.1 mm sodium vanadate, and 1% Triton X-100. Lysates were subjected to immunoprecipitation with anti-β4, anti-FAK, anti-Met, anti-Src, and anti-Grb2 antibodies (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Immunoprecipitates were separated by SDS-PAGE (7–12% polyacrylamide), blotted to nitrocellulose membranes, and probed with anti-β4, anti-FAK, anti-Src, anti-Grb2, as well as anti-pY antibodies. The ERK activity was determined from lysates of B16-F10 transfectants bound to mCLCA1- or PLL-coated dishes (see above) using anti-ERK antibody-conjugated agarose beads, followed by in vitrokinase assay (29Mainiero F. Pepe A. Wary K.K. Spinardi L. Mohammadi M. Schlessinger J. Giancotti F.G. EMBO J. 1995; 14: 4470-4481Crossref PubMed Scopus (235) Google Scholar). In brief, the kinase reaction was initiated by adding to the beads 25 μl of kinase buffer (25 mmTris-HCl, pH 7.5, 12.5 mm β-glycerophosphate, 7.5 mm MgCl2, 20 μm cold ATP, 0.5 mm sodium orthovanadate) containing 5 μCi of [γ-32P]ATP (ICN Biochemicals, Irvine, CA) and 2.5 μg of myelin basic protein (MBP). After 30 min of incubation at 30 °C, samples were boiled and separated by SDS-PAGE, and pMBP was visualized by autoradiography and quantified by Cerenkov counting. The BrdUrd assay was performed by a modification of the procedure by Brunet et al. (30Brunet A. Roux D. Lenormand P. Dowd S. Keyse S. Pouysseágur J. EMBO J. 1999; 18: 664-674Crossref PubMed Scopus (521) Google Scholar). In brief, the day after co-transfection with GFP and either wtFAK, FRNK, FAKY397F, or vector (mock), B16-F10 cells were serum-starved for 24 h, then seeded into wells (1 × 104 cells/well) of Costar 96-well assay plates (Corning, Corning, NY) coated with either mCLCA1 (3 μg/ml), fibronectin (5 μg/ml), or PLL (20 μg/ml) in the presence of DMEM containing 0.5% bovine calf serum and 100 μm 5-BrdUrd. After 16 h of incubation at 37 °C, cells were washed with DMEM, and the plates were mounted on the stage of an Olympus IX70 fluorescent microscope. Using a 40× objective, 20 fields (5/well) were recorded for each transfectant carefully registering the stage coordinates for each field. Cells were then fixed with methanol:formaldehyde (99:1, v/v) for 15 min at −20 °C followed by 3.7% formaldehyde in PBS for 15 min at room temperature. After rinses in PBS, chromatin was rendered accessible by a 10-min treatment with 2 m HCl, completely abolishing the GFP fluorescence. Cells were thoroughly washed with PBS, blocked with PBS/1% BSA/goat anti-mouse IgG (1:100) for 30 min at 37 °C, then incubated with anti-BrdUrd mAb (1:100) in PBS/1% BSA for 30 min at 37 °C. The secondary antibody was fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:500 in PBS/1% BSA, 30 min, 37 °C) (ICN). The fields recorded for GFP(+) cells were then rephotographed, and cells were scored for anti-BrdUrd staining. The percent BrdUrd(+) cells per GFP(+) cells was determined (a minimum of 200–300 green cells were evaluated per transfectant). Serum-starved B16-F10 cells co-transfected with GFP and wtFAK, FRNK, FAKY397F, or vector alone were seeded at a concentration of 200 green fluorescent cells/well into 96-well microtitration plates (Corning) coated with mCLCA1 (∼3 μg/ml, overnight, 4 °C). The mean number of green fluorescent cells was counted 48 h after seeding (96 h after transfection) for each transfectant and expressed as the percentage of GFP(+)cells in mock transfected B16-F10 cells. For tumor cell growth on endothelial cell monolayers, lung matrix-modulated BAEC (5 × 104/cm2) (5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 24Pauli B.U. Lee C.L. Lab. Invest. 1988; 58: 379-387PubMed Google Scholar) were seeded into 48-well plates and grown to confluence, then seeded with 3 × 103 B16-F10 cells co-transfected with GFP/wtFAK or GFP/FRNK. Tumor cells were incubated on the BAEC monolayers in DMEM + 1% FBS for 24, 48, 72, 96, and 120 h at 37 °C. Ten random areas (100×) were photographed daily for each transfectant, the number of GFP(+) cells per field was counted, and the counts were averaged. Adhesion and lung colony assays were performed as described previously (3Abdel-Ghany M. Cheng H.-C. Elble R.C. Pauli B.U. J. Biol. Chem. 2001; 276: 25438-25446Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, 5Zhu D.Z. Cheng C.F. Pauli B.U. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9568-9572Crossref PubMed Scopus (124) Google Scholar, 6Zhu D. Cheng C.F. Pauli B.U. J. Clin. Invest. 1992; 89: 1718-1724Crossref PubMed Scopus (51) Google Scholar). To determine the effects of wt and mutant FAK on the lung colony efficiency, B16-F10 were injected into the lateral tail vein of 6-week-old male C57BL/6 mice (eight mice/experiment) 48 h after transient transfection with FRNK, FAKY397F, or vector alone. Mice were sacrificed 21 days after tumor cell injection. Median and range of the number of lung colonies and mean ± S.D. of the lung weights were determined for each transfectant. Previous studies in our laboratory showed that intravenous co-injection of the anti-bCLCA2 (Lu-ECAM-1) mAb6D3 and [125I]iododeoxyuridine-labeled B16-F10 cells into syngeneic mice caused an almost complete clearance of tumor cells from mouse lungs within 3–5 days and a concomitant dramatic reduction in the number of lung metastases (6Zhu D. Cheng C.F. Pauli B.U. J. Clin. Invest. 1992; 89: 1718-1724Crossref PubMed Scopus (51) Google Scholar) (Fig.1A). In contrast, co-injection with mIgG resulted in incomplete tumor cell clearance and large numbers of metastases (Fig. 1A). These observations strongly suggested that mouse pulmonary endothelia express a CLCA family member that facilitates lung colonization by B16-F10 cells. To explore this possibility, we performed RT-PCR on RNA isolated from mouse lungs and cultured mouse pulmonary endothelial cells using degenerate primers based on the bCLCA2 amino acid sequence. A 414-bp PCR product was amplified from these sources in addition to RNA from mouse aortic endothelial cells, but not B16-F10 and HEK293 cells (Fig.1B). Sequencing of individual PCR products revealed 100% identity with the previously cloned mCLCA1 (15Gandhi R. Elble R.C. Gruber A.D. Schreur K.D., Ji, H.L. Fuller C.M. Pauli B.U. J. Biol. Chem. 1998; 273: 32096-32101Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The mCLCA1 protein products were subsequently isolated from extracts of mouse lungs, whose endothelia had been surface-biotinylated by in situ vascular perfusion and identified by Western blotting using streptavidin-HRP and anti-bCLCA2 pAb(rat4), respectively (Fig. 1C). Protein processing of wt mCLCA1 was identical to that of recombinant mCLCA1 extracted and immunopurified from mCLCA1-transfected HEK293 cells, yielding a 125-kDa precursor protein and 90- and 35-kDa proteolytic processing products (15Gandhi R. Elble R.C. Gruber A.D. Schreur K.D., Ji, H.L. Fuller C.M. Pauli B.U. J. Biol. Chem. 1998; 273: 32096-32101Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 23Elble R. Widom J. Gruber A.D. Abdel-Ghany M. Levine R. Goodwin A. Cheng H.-C. Pauli B.U. J. Biol. Chem. 1997; 272: 27853-27861Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Both wt and recombinant mCLCA1 supported strong B16-F10 adhesion that was inhibited selectively by mAb6D3 (Fig.1D). To determine whether endothelial mC" @default.
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