An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10, Mig, and I-TAC, and acts as functional receptor for platelet factor 4
Laura Lasagni, Michela Francalanci, Francesco Annunziato, Elena Lazzeri, Stefano Giannini, Lorenzo Cosmi, Costanza Sagrinati, Benedetta Mazzinghi, Claudio Orlando, Enrico Maggi, Fabio Marra, Sergio Romagnani, Mario Serio, Paola Romagnani, Laura Lasagni, Michela Francalanci, Francesco Annunziato, Elena Lazzeri, Stefano Giannini, Lorenzo Cosmi, Costanza Sagrinati, Benedetta Mazzinghi, Claudio Orlando, Enrico Maggi, Fabio Marra, Sergio Romagnani, Mario Serio, Paola Romagnani
Abstract
The chemokines CXCL9/Mig, CXCL10/IP-10, and CXCL11/I-TAC regulate lymphocyte chemotaxis, mediate vascular pericyte proliferation, and act as angiostatic agents, thus inhibiting tumor growth. These multiple activities are apparently mediated by a unique G protein-coupled receptor, termed CXCR3. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, including a powerful angiostatic effect, but its specific receptor is still unknown. Here, we describe a distinct, previously unrecognized receptor named CXCR3-B, derived from an alternative splicing of the CXCR3 gene that mediates the angiostatic activity of CXCR3 ligands and also acts as functional receptor for CXCL4. Human microvascular endothelial cell line-1 (HMEC-1), transfected with either the known CXCR3 (renamed CXCR3-A) or CXCR3-B, bound CXCL9, CXCL10, and CXCL11, whereas CXCL4 showed high affinity only for CXCR3-B. Overexpression of CXCR3-A induced an increase of survival, whereas overexpression of CXCR3-B dramatically reduced DNA synthesis and up-regulated apoptotic HMEC-1 death through activation of distinct signal transduction pathways. Remarkably, primary cultures of human microvascular endothelial cells, whose growth is inhibited by CXCL9, CXCL10, CXCL11, and CXCL4, expressed CXCR3-B, but not CXCR3-A. Finally, monoclonal antibodies raised to selectively recognize CXCR3-B reacted with endothelial cells from neoplastic tissues, providing evidence that CXCR3-B is also expressed in vivo and may account for the angiostatic effects of CXC chemokines.
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References
- Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity. 12:121–127.
- Rossi, D., and A. Zlotnik. 2000. The biology of chemokines and their receptors. Annu. Rev. Immunol. 18:217–242.
- Grone, H.J., C.D. Cohen, E. Grone, C. Schmidt, M. Kretzler, D. Schlondorff, and P.J. Nelson. 2002. Spatial and temporally restricted expression of chemokines and chemokine receptors in the developing human kidney. J. Am. Soc. Nephrol. 13:957–967.
- Belperio, J.A., M.P. Keane, D.A. Arenberg, C.L. Addison, J.E. Ehlert, M.D. Burdick, and R.M. Strieter. 2000. CXC chemokines in angiogenesis. J. Leukoc. Biol. 68:1–8.
- Homey, B., A. Müller, and A. Zlotnik. 2002. Chemokines: agents for the immunotherapy of cancer? Nat. Rev. Immunol. 2:175–184.
- Strieter, R.M., J.A. Belperio, and M.P. Keane. 2002. CXC chemokines in angiogenesis related to pulmonary fibrosis. Chest. 122:298S–301S.
- Arenberg, D.A., S.L. Kunkel, P.J. Polverini, S.B. Morris, M.D. Burdick, M.C. Glass, D.T. Taub, M.D. Iannettoni, R.I. Whyte, and R.M. Strieter. 1996. Interferon-γ–inducible protein 10 (IP-10) is an angiostatic factor that inhibits human non-small cell lung cancer (NSCLC) tumorigenesis and spontaneous metastases. J. Exp. Med. 184:981–992.
- Sgadari, C., J.M. Farber, A.L. Angiolillo, F. Liao, J. Teruya-Feldstein, P.R. Burd, L. Yao, G. Gupta, C. Kanegane, and G. Tosato. 1997. Mig, the monokine induced by interferon-gamma, promotes tumor necrosis in vivo. Blood. 89:2635–2643.
- Tanaka, T., Y. Manome, P. Wen, D.W. Kufe, and H.A. Fine. 1997. Viral vector-mediated transduction of a modified platelet factor 4 cDNA inhibits angiogenesis and tumor growth. Nat. Med. 3:437–442.
- Maione, T.E., G.S. Gray, J. Petro, A.J. Hunt, A.L. Donner, S.I. Bauer, H.F. Carson, and R.J. Sharpe. 1990. Inhibition of angiogenesis by recombinant human platelet factor-4 and related peptides. Science. 247:77–79.
- Romagnani, P., F. Annunziato, L. Lasagni, E. Lazzeri, C. Beltrame, M. Francalanci, M. Uguccioni, G. Galli, L. Cosmi, L. Maurenzig, et al. 2001. Cell cycle-dependent expression of CXC chemokine receptor 3 by endothelial cells mediates angiostatic activity. J. Clin. Invest. 107:53–63.
- Salcedo, R., J.H. Resau, D. Halverson, E.A. Hudson, M. Dambach, D. Powell, K. Wasserman, and J.J. Oppenheim. 2000. Differential expression and responsiveness of chemokine receptors (CXCR1-3) by human microvascular endothelial cells and umbilical vein endothelial cells. FASEB J. 14:2055–2064.
- Arenberg, D.A., A. Zlotnick, S.R. Strom, M.D. Burdick, and R.M. Strieter. 1999. The murine CC chemokine, 6C-kine, inhibits tumor growth and angiogenesis in a human lung cancer SCID mouse model. Cancer Immunol. Immunother. 49:587–592.
- Luster, A.D., S.M. Greenberg, and P. Leder. 1995. The IP-10 chemokine binds to a specific cell surface heparan sulfate site shared with platelet factor 4 and inhibits endothelial cell proliferation. J. Exp. Med. 182:219–231.
- Strieter, R.M., P.J. Polverini, S.L. Kunkel, D.A. Arenberg, M.D. Burdick, J. Kasper, J. Dzuiba, J. Van Damme, A. Walz, D. Marriott, et al. 1995. The functional role of the ELR motif in CXC chemokine-mediated angiogenesis. J. Biol. Chem. 270:27348–27357.
- Aronica, S.M., C. Mantel, R. Gonin, M.S. Marshall, A. Sarris, S. Cooper, N. Hague, X.F. Zhang, and H.E. Broxmeyer. 1995. Interferon-inducible protein 10 and macrophage inflammatory protein-1 alpha inhibit growth factor stimulation of Raf-1 kinase activity and protein synthesis in a human growth factor-dependent hematopoietic cell line. J. Biol. Chem. 270:21998–22007.
- Bonecchi, R., G. Bianchi, P.P. Bordignon, D. D'Ambrosio, R. Lang, A. Borsetti, S. Sozzani, P. Allavena, P.A. Gray, A. Mantovani, et al. 1998. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J. Exp. Med. 187:129–134.
- Sallusto, F., D. Lenig, C.R. Mackay, and A. Lanzavecchia. 1998. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J. Exp. Med. 187:875–883.
- Loetscher, M., B. Gerber, P. Loetscher, S.A. Jones, L. Piali, I. Clark-Lewis, M. Baggiolini, and B. Moser. 1996. Chemokine receptor specific for IP-10 and Mig: structure, function, and expression in activated T lymphocytes. J. Exp. Med. 184:963–969.
- Janatpour, M.J., S. Hudak, M. Sathe, J.D. Sedgwick, and L.M. McEvoy. 2001. Tumor necrosis factor–dependent segmental control of MIG expression by high endothelial venules in inflamed lymph nodes regulates monocyte recruitment. J. Exp. Med. 194:1375–1384.
- Penna, G., S. Sozzani, and L. Adorini. 2001. Cutting edge: selective usage of chemokine receptors by plasmacytoid dendritic cells. J. Immunol. 167:1862–1866.
- Romagnani, P., C. Beltrame, F. Annunziato, L. Lasagni, M. Luconi, G. Galli, L. Cosmi, E. Maggi, M. Salvadori, C. Pupilli, et al. 1999. Role for interactions between IP-10/Mig and their receptor (CXCR3) in proliferative glomerulonephritis. J. Am. Soc. Nephrol. 10:2518–2526.
- Romagnani, P., E. Lazzeri, L. Lasagni, C. Mavilia, C. Beltrame, M. Francalanci, M. Rotondi, F. Annunziato, L. Maurenzig, L. Cosmi, et al. 2002. IP-10 and Mig production by glomerular cells in human proliferative glomerulonephritis and regulation by nitric oxide. J. Am. Soc. Nephrol. 13:53–64.
- Bonacchi, A., P. Romagnani, R.G. Romanelli, E. Efsen, F. Annunziato, L. Lasagni, M. Francalanci, M. Serio, G. Laffi, M. Pinzani, et al. 2001. Signal transduction by the chemokine receptor CXCR3: activation of Ras/ERK, Src, and phosphatidylinositol 3-kinase/Akt controls cell migration and proliferation in human vascular pericytes. J. Biol. Chem. 276:9945–9954.
- Pupilli, C., L. Lasagni, P. Romagnani, F. Bellini, M. Mannelli, N. Misciglia, C. Mavilia, U. Vellei, D. Villari, and M. Serio. 1999. Angiotensin II stimulates the synthesis and secretion of vascular permeability factor/vascular endothelial growth factor in human mesangial cells. J. Am. Soc. Nephrol. 10:245–255.
- Romagnani, P., M. Rotondi, E. Lazzeri, L. Lasagni, M. Francalanci, A. Buonamano, S. Milani, P. Vitti, L. Chiovato, M. Tonacchera, et al. 2002. Expression of IP-10/CXCL10 and MIG/CXCL9 in the thyroid and increased levels of IP-10/CXCL10 in the serum of patients with recent-onset Graves' disease. Am. J. Pathol. 161:195–206.
- Jenh, C.H., M.A. Cox, H. Kaminski, M. Zhang, H. Byrnes, J. Fine, D. Lundell, C.C. Chou, S.K. Narula, and P.J. Zavodny. 1999. Cutting edge: species specificity of the CC chemokine 6Ckine signaling through the CXC chemokine receptor CXCR3: human 6Ckine is not a ligand for the human or mouse CXCR3 receptors. J. Immunol. 162:3765–3769.
- Ades, E.W., F.J. Candal, R.A. Swerlick, V.G. George, S. Summers, D.C. Bosse, and T.J. Lawley. 1992. HMEC-1: establishment of an immortalized human microvascular endothelial cell line. J. Invest. Dermatol. 99:683–690.
- Gengrinovitch, S., S.M. Greenberg, T. Cohen, H. Gitay-Goren, P. Rockwell, T.E. Maione, B.Z. Levi, and G. Neufeld. 1995. Platelet factor-4 inhibits the mitogenic activity of VEGF121 and VEGF165 using several concurrent mechanisms. J. Biol. Chem. 270:15059–15065.
- Pugliese, G., F. Pricci, G. Romeo, F. Pugliese, P. Mene, S. Giannini, B. Cresci, G. Galli, C.M. Rotella, H. Vlassara, et al. 1997. Upregulation of mesangial growth factor and extracellular matrix synthesis by advanced glycation end products via a receptor-mediated mechanism. Diabetes. 46:1881–1887.
- De Lean, A., P.J. Munson, and D. Rodbard. 1978. Simultaneous analysis of families of sigmoidal curves: application to bioassay, radioligand assay, and physiological dose-response curves. Am. J. Physiol. 235:E97–E102.
- Van Riper, G., S. Siciliano, P.A. Fischer, R. Meurer, M.S. Springer, and H. Rosen. 1993. Characterization and species distribution of high affinity GTP-coupled receptors for human RANTES and monocyte chemoattractant protein 1. J. Exp. Med. 177:851–856.
- Chan, A.I., and P.C. Keng. 1987. Potentiation of radiation cytotoxicity by recombinant interferons, a phenomenon associated with increased blockage at the G2-M phase of the cell cycle. Cancer Res. 47:4338–4341.
- Romagnani, P., F. Annunziato, R. Manetti, C. Mavilia, L. Lasagni, C. Manuelli, G.B. Vannelli, V. Vanini, E. Maggi, C. Pupilli, et al. 1998. High CD30 ligand expression by epithelial cells and Hassal's corpuscles in the medulla of human thymus. Blood. 91:3323–3332.
- Files, J.C., T.W. Malpass, E.K. Yee, J.L. Ritchie, and L.A. Harker. 1981. Studies of human platelet α-release in vivo. Blood. 58:607–618.
- Perollet, C., Z.C. Han, C. Savona, J.B. Caen, and A. Bikfalvi. 1998. Platelet factor 4 modulates fibroblast growth factor 2 (FGF-2) activity and inhibits FGF-2 dimerization. Blood. 91:3289–3299.
- Sulpice, E., M. Bryckaert, J. Lacour, J.O. Contreres, and G. Tobelem. 2002. Platelet factor 4 inhibits FGF2-induced endothelial cell proliferation via the extracellular signal-regulated kinase pathway but not by the phosphatidylinositol 3-kinase pathway. Blood. 100:3087–3094.
- Gentilini, G., N.C. Kirschbaum, J.A. Augustine, R.H. Aster, and G.P. Visenti. 1999. Inhibition of human umbilical vein endothelial cell proliferation by the CXC chemokine, platelet factor 4 (PF-4), is associated with impaired downregulation of p21Cip1/WAF1. Blood. 93:25–33.
- Shiraha, H., A. Glading, K. Gupta, and A. Wells. 1999. IP-10 inhibits epidermal growth factor–induced motility by decreasing epidermal growth factor receptor–mediated calpain activity. J. Cell Biol. 146:243–254.
- Maione, T.E., G.S. Gray, A.J. Hunt, and R.J. Sharpe. 1991. Inhibition of tumor growth in mice by an analogue of platelet factor 4 that lacks affinity for heparin and retains potent angiostatic activity. Cancer Res. 51:2077–2083.
- Lecomte-Raclet, L., M. Alemany, A. Sequeira-Le Grand, J. Amiral, G. Quentin, A.M. Vissac, J.P. Caen, and Z.C. Han. 1998. New insights into the negative regulation of hematopoiesis by chemokine platelet factor 4 and related peptides. Blood. 91:2772–2780.
- Jouan, V., X. Canron, M. Alemany, J.P. Caen, G. Quentin, J. Plouet, and A. Bikfalvi. 1999. Inhibition of in vitro angiogenesis by platelet factor-4-derived peptides and mechanism of action. Blood. 94:984–993.
- Hansell, P., T.E. Maione, and P. Borgstrom. 1995. Selective binding of platelet factor 4 to regions of active angiogenesis in vivo. Am. J. Physiol. 269:H829–H836.
- Soto, H., W. Wang, R.M. Strieter, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, J. Hedrick, and A. Zlotnik. 1998. The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3. Proc. Natl. Acad. Sci. USA. 95:8205–8210.
- Rappert, A., K. Biber, C. Nolte, M. Lipp, A. Schubel, B. Lu, N.P. Gerard, C. Gerard, H.W. Boddeke, and H. Kettenmann. 2002. Secondary lymphoid tissue chemokine (CCL21) activates CXCR3 to trigger a Cl-current and chemotaxis in murine microglia. J. Immunol. 168:3221–3226.
- Maghazachi, A.A., B.S. Skalhegg, B. Rolstad, and A. Al-Aoukaty. 1997. Interferon-inducible protein-10 and lymphotactin induce the chemotaxis and mobilization of intracellular calcium in natural killer cells through pertussis toxin-sensitive and -insensitive heterotrimeric G-proteins. FASEB J. 11:765–774.
- Marinissen, M.J., and J.S. Gutkind. 2001. G-protein-coupled receptors and signaling networks: emerging paradigms. Trends Pharmacol. Sci. 22:368–376.
- Kim, S., M. Bakre, H. Yin, and J.A. Varner. 2002. Inhibition of endothelial cell survival and angiogenesis by protein kinase A. J. Clin. Invest. 110:933–941.
- Ho, H.H., D. Du, and M.C. Gershengorn. 1999. The N terminus of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor is necessary for high affinity chemokine binding but not for constitutive activity. J. Biol. Chem. 274:31327–31332.
- Couty, J.P., E. Geras-Raaka, B.B. Weksler, and M.C. Gershengorn. 2001. Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor signals through multiple pathways in endothelial cells. J. Biol. Chem. 276:33805–33811.
- Zhou, Y., S. Wang, B.G. Yue, A. Gobl, and K. Oberg. 2002. Effects of interferon alpha on the expression of p21cip1/waf1 and cell cycle distribution in carcinoid tumors. Cancer Invest. 20:348–356.
- Lee, T.H., L.Y. Chuang, and W.C. Hung. 2000. Induction of p21WAF1 expression via Sp1-binding sites by tamoxifen in estrogen receptor-negative lung cancer cells. Oncogene. 19:3766–3773.
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