ANGPTL3 Stimulates Endothelial Cell Adhesion and Migration via Integrin αvβ3 and Induces Blood Vessel Formation in Vivo
2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês
10.1074/jbc.m109768200
ISSN1083-351X
AutoresGieri Camenisch, M. Teresa Pisabarro, Daniel Sherman, Joe Kowalski, Mark Nagel, Phil Hass, Ming-Hong Xie, Austin Gurney, Sarah Bodary, Xiao Liang, Kevin Clark, Maureen H. Beresini, Napoleone Ferrara, Hans‐Peter Gerber,
Tópico(s)Caveolin-1 and cellular processes
ResumoThe angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to αvβ3 and induced integrin αvβ3-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin αvβ3 and suggest a possible role in the regulation of angiogenesis. The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to αvβ3 and induced integrin αvβ3-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin αvβ3 and suggest a possible role in the regulation of angiogenesis. The growth of new blood vessels is a prerequisite during normal physiological processes of embryonic and postnatal development. However, proliferation of new blood vessels from preexisting capillaries, a process termed angiogenesis, also plays a key role in the progression of solid tumor growth, diabetic retinopathies, psoriasis, inflammation, and rheumatoid arthritis (1.Ferrara N. Rec. Prog. Horm. Res. 2000; 55: 15-36PubMed Google Scholar). Angiogenesis not only is dependent on secreted factors like VEGF 1The abbreviations used are: VEGFvascular endothelial growth factorBSAbovine serum albuminPMAphorbol 12-myristate 13-acetateFAKfocal adhesion kinaseHUVEChuman umbilical venous endothelial cellHMVEChuman microvascular vein endothelial cellCS-CCell System completePBSphosphate-buffered salineCHOChinese hamster ovaryFBNfibrinogenARP1angiopoietin-related protein 1VNvitronectinAng1 and -2angiopoietin 1 and 2, respectivelyMAPKmitogen-activated protein kinasehANGPTL3human ANGPTL3mANGPTL3murine ANGPTL31The abbreviations used are: VEGFvascular endothelial growth factorBSAbovine serum albuminPMAphorbol 12-myristate 13-acetateFAKfocal adhesion kinaseHUVEChuman umbilical venous endothelial cellHMVEChuman microvascular vein endothelial cellCS-CCell System completePBSphosphate-buffered salineCHOChinese hamster ovaryFBNfibrinogenARP1angiopoietin-related protein 1VNvitronectinAng1 and -2angiopoietin 1 and 2, respectivelyMAPKmitogen-activated protein kinasehANGPTL3human ANGPTL3mANGPTL3murine ANGPTL3 or the angiopoietins, which bind to and ligate their respective tyrosine kinase receptors expressed on endothelial cells, but is also influenced by cell adhesion molecules binding to their ligands present within the extracellular matrix (2.Byzova V.T. Goldman K.C. Pampori N. Thomas A.K. Bett A. Shattil J.S. Plow F.E. Mol. Cell. 2000; 6: 851-860Abstract Full Text Full Text PDF PubMed Google Scholar). Inactivation of various genes encoding specific cell adhesion molecules or administration of function blocking antibodies targeting cell adhesion molecules in various animal models resulted in profound inhibitory effects on the angiogenic response of endothelial cells (3.Eliceiri B.P. Cheresh D.A. Mol. Med. 1998; 4: 741-750Crossref PubMed Google Scholar).The integrin family of cell adhesion molecules are two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. This family is composed of over 15 α and eight β subunits expressed in at least 22 different αβ heterodimeric combinations. Among these, a combination of six (αvβ3, αvβ5, α5β1, α2β1, αvβ1, and α1β1) has been implicated in angiogenesis (4.Hynes R.O. Bader B.L. Thromb. Haemostasis. 1997; 78: 83-87Crossref PubMed Scopus (58) Google Scholar, 5.Hynes R.O. Bader B.L. Hodivala-Dilke K. Braz. J. Med. Biol. Res. 1999; 32: 501-510Crossref PubMed Scopus (106) Google Scholar). Integrins facilitate cellular adhesion to and migration on extracellular matrix proteins located within the intercellular spaces and basement membranes. Some integrins were shown to bind to FBN-like domains encoded by various ligands found within the extracellular matrix (6.Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 7.Yokoyama K. Erickson H.P. Ikeda Y. Takada Y. J. Biol. Chem. 2000; 275: 16891-16898Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Integrin αvβ3 binds to a wide variety of extracellular matrix proteins including vitronectin, fibronectin, fibrinogen, laminin, collagen, von Willebrand factor, osteopontin, and a fragment of MMP2 (PEX) among others (for a review, see Ref. 8.Eliceiri B.P. Cheresh D.A. Cancer J. Sci. Am. 2000; 6 Suppl. 3: 245-249Google Scholar). Despite its promiscuous ligand binding behavior, αvβ3 is not widely expressed in adult tissues and was found on some vascular, intestinal, and uterine smooth muscle cells (9.Brem R.B. Robbins S.G. Wilson D.J. O'Rourke L.M. Mixon R.N. Robertson J.E. Planck S.R. Rosenbaum J.T. Invest. Ophthalmol. Vis. Sci. 1994; 35: 3466-3474PubMed Google Scholar). This receptor was also found on activated leukocytes, on macrophages and osteoclasts, where it regulates bone resorption (10.McHugh K.P. Hodivala-Dilke K. Zheng M.H. Namba N. Lam J. Novack D. Feng X. Ross F.P. Hynes R.O. Teitelbaum S.L. J. Clin. Invest. 2000; 105: 433-440Crossref PubMed Scopus (573) Google Scholar). Most prominently, αvβ3 becomes up-regulated on endothelial cells exposed to hypoxia and cytokines such as VEGF-A (11.Walton H.L. Corjay M.H. Mohamed S.N. Mousa S.A. Santomenna L.D. Reilly T.M. J. Cell. Biochem. 2000; 78: 674-680Crossref PubMed Scopus (38) Google Scholar, 12.Suzuma K. Takagi H. Otani A. Honda Y. Invest. Ophthalmol. Vis. Sci. 1998; 39: 1028-1035PubMed Google Scholar) and was found to be overexpressed on tumor vasculature or in atherosclerotic arteries (13.Hoshiga M. Alpers C.E. Smith L.L. Giachelli C.M. Schwartz S.M. Circ. Res. 1995; 77: 1129-1135Crossref PubMed Scopus (210) Google Scholar).ANGPTL3 was previously found to be expressed in a liver-specific manner during development and in adults (14.Conklin D. Gilbertson D. Taft D.W. Maurer M.F. Whitmore T.E. Smith D.L. Walker K.M. Chen L.H. Wattler S. Nehls M. Lewis K.B. Genomics. 1999; 62: 477-482Crossref PubMed Scopus (121) Google Scholar), and more recently, it was found to be involved in the regulation of serum lipid levels in mice (15.Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Nat. Genet. 2002; 2: 151-157Crossref Scopus (314) Google Scholar). We independently cloned ANGPTL3 based on sequence homologies with the FBN-like domains located within the carboxyl terminus of the angiopoietins (16.Procopio W.N. Pelavin P.I. Lee W.M. Yeilding N.M. J. Biol. Chem. 1999; 274: 30196-30201Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Since ANGPTL3 did not bind to the angiopoietin receptor Tie2, we tried to identify potential candidate receptors by taking advantage of the structural information available for other, related FBN-like domains. The three-dimensional structure of the C terminus of the γ-chain within human fibrinogen has been solved by x-ray crystallography (17.Yee V.C. Pratt K.P. Cote H.C. Trong I.L. Chung D.W. Davie E.W. Stenkamp R.E. Teller D.C. Structure. 1997; 5: 125-138Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), and we have used this structure information as a template to build a three-dimensional model of the FBN-like domain of ANGPTL3. This model strongly suggested members of the integrin family of cell adhesion molecules as potential candidates for binding. Cell-based assays with recombinant proteins and direct protein-binding experiments revealed that ANGPTL3 was binding to αvβ3 and induced endothelial cell migration and adhesion, which was potently abolished by the presence of function blocking antibody-targeting integrin αvβ3. The robust induction of blood vessel growth by ANGPTL3 in the rat corneal angiogenesis assay revealed that this liver-specific, secreted protein is a novel angiogenic factor. The growth of new blood vessels is a prerequisite during normal physiological processes of embryonic and postnatal development. However, proliferation of new blood vessels from preexisting capillaries, a process termed angiogenesis, also plays a key role in the progression of solid tumor growth, diabetic retinopathies, psoriasis, inflammation, and rheumatoid arthritis (1.Ferrara N. Rec. Prog. Horm. Res. 2000; 55: 15-36PubMed Google Scholar). Angiogenesis not only is dependent on secreted factors like VEGF 1The abbreviations used are: VEGFvascular endothelial growth factorBSAbovine serum albuminPMAphorbol 12-myristate 13-acetateFAKfocal adhesion kinaseHUVEChuman umbilical venous endothelial cellHMVEChuman microvascular vein endothelial cellCS-CCell System completePBSphosphate-buffered salineCHOChinese hamster ovaryFBNfibrinogenARP1angiopoietin-related protein 1VNvitronectinAng1 and -2angiopoietin 1 and 2, respectivelyMAPKmitogen-activated protein kinasehANGPTL3human ANGPTL3mANGPTL3murine ANGPTL31The abbreviations used are: VEGFvascular endothelial growth factorBSAbovine serum albuminPMAphorbol 12-myristate 13-acetateFAKfocal adhesion kinaseHUVEChuman umbilical venous endothelial cellHMVEChuman microvascular vein endothelial cellCS-CCell System completePBSphosphate-buffered salineCHOChinese hamster ovaryFBNfibrinogenARP1angiopoietin-related protein 1VNvitronectinAng1 and -2angiopoietin 1 and 2, respectivelyMAPKmitogen-activated protein kinasehANGPTL3human ANGPTL3mANGPTL3murine ANGPTL3 or the angiopoietins, which bind to and ligate their respective tyrosine kinase receptors expressed on endothelial cells, but is also influenced by cell adhesion molecules binding to their ligands present within the extracellular matrix (2.Byzova V.T. Goldman K.C. Pampori N. Thomas A.K. Bett A. Shattil J.S. Plow F.E. Mol. Cell. 2000; 6: 851-860Abstract Full Text Full Text PDF PubMed Google Scholar). Inactivation of various genes encoding specific cell adhesion molecules or administration of function blocking antibodies targeting cell adhesion molecules in various animal models resulted in profound inhibitory effects on the angiogenic response of endothelial cells (3.Eliceiri B.P. Cheresh D.A. Mol. Med. 1998; 4: 741-750Crossref PubMed Google Scholar). vascular endothelial growth factor bovine serum albumin phorbol 12-myristate 13-acetate focal adhesion kinase human umbilical venous endothelial cell human microvascular vein endothelial cell Cell System complete phosphate-buffered saline Chinese hamster ovary fibrinogen angiopoietin-related protein 1 vitronectin angiopoietin 1 and 2, respectively mitogen-activated protein kinase human ANGPTL3 murine ANGPTL3 vascular endothelial growth factor bovine serum albumin phorbol 12-myristate 13-acetate focal adhesion kinase human umbilical venous endothelial cell human microvascular vein endothelial cell Cell System complete phosphate-buffered saline Chinese hamster ovary fibrinogen angiopoietin-related protein 1 vitronectin angiopoietin 1 and 2, respectively mitogen-activated protein kinase human ANGPTL3 murine ANGPTL3 The integrin family of cell adhesion molecules are two-way signaling receptors responsible for the attachment of cells to the extracellular matrix and for cell-cell interactions that underlie immune responses, tumor metastasis, and progression of atherosclerosis and thrombosis. This family is composed of over 15 α and eight β subunits expressed in at least 22 different αβ heterodimeric combinations. Among these, a combination of six (αvβ3, αvβ5, α5β1, α2β1, αvβ1, and α1β1) has been implicated in angiogenesis (4.Hynes R.O. Bader B.L. Thromb. Haemostasis. 1997; 78: 83-87Crossref PubMed Scopus (58) Google Scholar, 5.Hynes R.O. Bader B.L. Hodivala-Dilke K. Braz. J. Med. Biol. Res. 1999; 32: 501-510Crossref PubMed Scopus (106) Google Scholar). Integrins facilitate cellular adhesion to and migration on extracellular matrix proteins located within the intercellular spaces and basement membranes. Some integrins were shown to bind to FBN-like domains encoded by various ligands found within the extracellular matrix (6.Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 7.Yokoyama K. Erickson H.P. Ikeda Y. Takada Y. J. Biol. Chem. 2000; 275: 16891-16898Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Integrin αvβ3 binds to a wide variety of extracellular matrix proteins including vitronectin, fibronectin, fibrinogen, laminin, collagen, von Willebrand factor, osteopontin, and a fragment of MMP2 (PEX) among others (for a review, see Ref. 8.Eliceiri B.P. Cheresh D.A. Cancer J. Sci. Am. 2000; 6 Suppl. 3: 245-249Google Scholar). Despite its promiscuous ligand binding behavior, αvβ3 is not widely expressed in adult tissues and was found on some vascular, intestinal, and uterine smooth muscle cells (9.Brem R.B. Robbins S.G. Wilson D.J. O'Rourke L.M. Mixon R.N. Robertson J.E. Planck S.R. Rosenbaum J.T. Invest. Ophthalmol. Vis. Sci. 1994; 35: 3466-3474PubMed Google Scholar). This receptor was also found on activated leukocytes, on macrophages and osteoclasts, where it regulates bone resorption (10.McHugh K.P. Hodivala-Dilke K. Zheng M.H. Namba N. Lam J. Novack D. Feng X. Ross F.P. Hynes R.O. Teitelbaum S.L. J. Clin. Invest. 2000; 105: 433-440Crossref PubMed Scopus (573) Google Scholar). Most prominently, αvβ3 becomes up-regulated on endothelial cells exposed to hypoxia and cytokines such as VEGF-A (11.Walton H.L. Corjay M.H. Mohamed S.N. Mousa S.A. Santomenna L.D. Reilly T.M. J. Cell. Biochem. 2000; 78: 674-680Crossref PubMed Scopus (38) Google Scholar, 12.Suzuma K. Takagi H. Otani A. Honda Y. Invest. Ophthalmol. Vis. Sci. 1998; 39: 1028-1035PubMed Google Scholar) and was found to be overexpressed on tumor vasculature or in atherosclerotic arteries (13.Hoshiga M. Alpers C.E. Smith L.L. Giachelli C.M. Schwartz S.M. Circ. Res. 1995; 77: 1129-1135Crossref PubMed Scopus (210) Google Scholar). ANGPTL3 was previously found to be expressed in a liver-specific manner during development and in adults (14.Conklin D. Gilbertson D. Taft D.W. Maurer M.F. Whitmore T.E. Smith D.L. Walker K.M. Chen L.H. Wattler S. Nehls M. Lewis K.B. Genomics. 1999; 62: 477-482Crossref PubMed Scopus (121) Google Scholar), and more recently, it was found to be involved in the regulation of serum lipid levels in mice (15.Koishi R. Ando Y. Ono M. Shimamura M. Yasumo H. Fujiwara T. Horikoshi H. Furukawa H. Nat. Genet. 2002; 2: 151-157Crossref Scopus (314) Google Scholar). We independently cloned ANGPTL3 based on sequence homologies with the FBN-like domains located within the carboxyl terminus of the angiopoietins (16.Procopio W.N. Pelavin P.I. Lee W.M. Yeilding N.M. J. Biol. Chem. 1999; 274: 30196-30201Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). Since ANGPTL3 did not bind to the angiopoietin receptor Tie2, we tried to identify potential candidate receptors by taking advantage of the structural information available for other, related FBN-like domains. The three-dimensional structure of the C terminus of the γ-chain within human fibrinogen has been solved by x-ray crystallography (17.Yee V.C. Pratt K.P. Cote H.C. Trong I.L. Chung D.W. Davie E.W. Stenkamp R.E. Teller D.C. Structure. 1997; 5: 125-138Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), and we have used this structure information as a template to build a three-dimensional model of the FBN-like domain of ANGPTL3. This model strongly suggested members of the integrin family of cell adhesion molecules as potential candidates for binding. Cell-based assays with recombinant proteins and direct protein-binding experiments revealed that ANGPTL3 was binding to αvβ3 and induced endothelial cell migration and adhesion, which was potently abolished by the presence of function blocking antibody-targeting integrin αvβ3. The robust induction of blood vessel growth by ANGPTL3 in the rat corneal angiogenesis assay revealed that this liver-specific, secreted protein is a novel angiogenic factor. We thank Tom Gadek for stimulating discussions and Minghong Yan and Karen O'Rourke for providing the leucine zipper and FLAG fusion vectors. We thank Peter Schow and Catherine Grimmer for help during FACS experiments and the sequencing group for excellent support. We thank everyone involved in the large scale CHO transient transfections, and we thank Jessica Foster for baculovirus expression and Michael Elliott for protein purification. We thank David Wood for excellent graphic assistance and everyone in the Ferrara laboratory for helpful discussions and support.
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