Regulation of <em>Notch1</em> and <em>Dll4</em> by Vascular Endothelial Growth Factor in Arterial Endothelial Cells: Implications for Modulating Arteriogenesis and Angiogenesis
Notch and its ligands play critical roles in cell fate determination. Expression of Notch and ligand in vascular endothelium and defects in vascular phenotypes of targeted mutants in the Notch pathway have suggested a critical role for Notch signaling in vasculogenesis and angiogenesis. However, the angiogenic signaling that controls Notch and ligand gene expression is unknown. We show here that vascular endothelial growth factor (VEGF) but not basic fibroblast growth factor can induce gene expression of Notch1 and its ligand, Delta-like 4 (Dll4), in human arterial endothelial cells. The VEGF-induced specific signaling is mediated through VEGF receptors 1 and 2 and is transmitted via the phosphatidylinositol 3-kinase/Akt pathway but is independent of mitogen-activated protein kinase and Src tyrosine kinase. Constitutive activation of Notch signaling stabilizes network formation of endothelial cells on Matrigel and enhances formation of vessel-like structures in a three-dimensional angiogenesis model, whereas blocking Notch signaling can partially inhibit network formation. This study provides the first evidence for regulation of Notch/Delta gene expression by an angiogenic growth factor and insight into the critical role of Notch signaling in arteriogenesis and angiogenesis.
Notch signaling is highly conserved through evolution and plays a fundamental role in the determination of cell fate (1, 48). It also affects cell cycle progression and apoptosis. In humans, there are four Notch receptors, Notch 1 to 4, and five ligands, including Jagged1 and -2 and Dll1, -3, and -4. Activation of Notch upon ligand binding is accompanied by proteolytic processing that releases an intracellular domain of Notch (NICD) from the membrane. The NICD then translocates into the nucleus and associates with the CSL [CBF-1 (RBP-Jκ)/Su(H)/Lag-1] family of DNA-binding proteins to form a transcriptional activator, which turns on transcription of a set of target genes, including the E(spl) (Enhancer of Split) group and others (28). Most of the Notch target genes encode transcription regulators, which in turn modulate cell fate by affecting the function of tissue-specific basic helix-loop-helix transcription factors or through other molecular targets, such as NF-κB (2).
Vasculogenesis and angiogenesis are processes of the formation of new vascular networks, which involve sprouting, branching, splitting, and differential growth of vessels from the primary plexus or existing vessel into a functioning circulation system (4, 10). Vessels develop into specific types, including arteries, veins, capillaries, and lymphatics. In adults, physiological angiogenesis occurs during the female reproductive cycle and in wound healing, while abnormal angiogenesis can be observed in solid tumor and rheumatoid arthritis. A number of cellular signaling pathways, such as vascular endothelial growth factor (VEGF) and its receptor (VEGFR), basic fibroblast growth factor (bFGF), transforming growth factor beta, and platelet-derived growth factor with their receptors, angiopoietin/Tie and ephrin/Eph, have been implicated in regulating vasculogenesis and angiogenesis (50). Among angiogenic regulators, VEGF family members VEGF-A (VEGF), -B, -C, -D, and -E and placenta growth factor and VEGFRs [VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1), and VEGFR3 (Flt-4)] are key mediators. VEGF stimulates vascular endothelial cells through VEGFR1 and VEGFR2, whereas VEGF-C and -D bind to VEGFR2 and VEGFR3 and primarily affect lymphangiogenesis (44).
Growing evidence suggests involvement of Notch signaling in the regulation of vascular formation. For instance, the human degenerative vascular disease cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) has been associated with mutations in Notch3 (16). In vertebrates, Notch1, Notch4, Jagged1, Jagged2, Dll1, and Dll4 are expressed in vascular endothelium (22, 25, 35, 42, 41, 46). Targeted Notch-1, Notch1/Notch 4, Jagged-1, and Dll-1 mutations all result in vascular defects (13, 15, 19, 49). Notch signaling must also be appropriately regulated in order to maintain normal vascular development, since expression of activated Notch4 in mouse embryonic endothelium results in vascular patterning defects (43). In zebrafish, development of the aorta requires the gridlock gene, a homologue of mammalian HES (Hairy/Enhancer of Split), which is regulated by Notch activation (51). Moreover, the essential roles of Jagged-1 and HES related 1 (HESR1) in modulating vessel formation in vitro have been well demonstrated (12, 53).
The precise role of Notch signaling in governing endothelial cell behavior remains unclear. In Notch1 and Notch1/Notch4 mouse embryos, the primary vascular plexus appeared to form normally, but embryos failed to remodel the plexus to form large and small blood vessels, indicating that Notch signaling is essential for angiogenic vascular morphogenesis and remodeling (15, 20). Notch signaling also plays a role in defining arterial endothelial cells and determining the formation of arteries through repression of venous fate. In zebrafish, DeltaC, a ligand for the Notch, is expressed in endothelial cells that contribute to the dorsal aorta but not the posterior cardinal vein at least 6 h before the onset of blood flow (36). The phenotype of the zebrafish gridlock gene mutant revealed a defect in the formation of the dorsal aorta but not in the vein (51). More direct evidence supporting the crucial role of gridlock in the control of the artery-vein decision in zebrafish embryos has been provided very recently (52). In mammals, Dll4, a newly identified ligand responsible for the activation of Notch1 and Notch4, is preferentially expressed in arterial endothelium (35), suggesting a potential role for Dll4 in modulating arterial development (arteriogenesis).
The angiogenic signaling pathways controlling Notch/Delta gene expression are unknown. The relationship between Notch signaling and other angiogenic regulators, such as VEGF, bFGF, transforming growth factor beta, platelet-derived growth factor, angiopoietin/Tie, and ephrin/Eph, has not been well investigated. Here we asked whether soluble angiogenic factors can regulate Notch/Delta gene expression and which specific signaling pathway delivers the initiation signal in human endothelial cells. Furthermore, the biological significance of expression of Notch1/Dll4 on endothelial cells has been addressed by inducing the activation of Notch signaling in arterial endothelial cells. Our findings provide the first example of regulation of Notch/Delta gene expression by a soluble growth factor and thus establish a functional linkage between two important angiogenic signaling pathways and also give insight into a critical role for Notch signaling in regulating arteriogenesis/angiogenesis.
We thank P. Carmeliet, D. W. Ball, and W. Ogawa for providing different recombinant adenoviruses; M. Detmar, M. Skobe, M. Shibuya, T. Kodach, and T. Honjo for various plasmids; and T. Sudo for anti-HES-1 antibody.
This work was supported by the McCabe Fund and grants from the National Institutes of Health (CA47159, CA25874, and CA10815).
- 1. Artavanis-Tsakonas, S., K. Matsuno, and M. E. Fortini. 1995. Notch signaling. Science268:225-232. [[PubMed]
- 2. Bellavia, D., A. F. Campese, E. Alesse, A. Vacca, M. P. Felli, A. Balestri, A. Stoppacciaro, C. Tiveron, L. Tatangelo, M. Giovarelli, C. Gaetano, L. Ruco, E. S. Hoffman, A. C. Hayday, U. Lendahl, L. Frati, A. Gulino, and I. Screpanti. 2000. Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in Notch3 transgenic mice. EMBO J.19:3337-3348.
- 3. Byzova, T. V., C. K. Goldman, N. Pampori, K. A. Thomas, A. Bett, S. J. Shattil, and E. F. Plow. 2000. A mechanism for modulation of cellular responses to VEGF: activation of the integrins. Mol. Cell6:851-860. [[PubMed]
- 4. Carmeliet, P. 2000. Mechanisms of angiogenesis and arteriogenesis. Nat. Med.6:389-395. [[PubMed]
- 5. Chung, C. N., Y. Hamaguchi, T. Honjo, and M. Kawaichi. 1994. Site-directed mutagenesis study on DNA binding regions of the mouse homologue of Suppressor of Hairless, RBP-Jκ. Nucleic Acids Res.22:2938-2944.
- 6. Cooper, M. T., and S. J. Bray. 1999. Frizzled regulation of Notch signaling polarizes cell fate in the Drosophila eye. Nature397:526-530. [[PubMed]
- 7. Dayanir, V., R. D. Meyer, K. Lashkari, and N. Rahimi. 2001. Identification of tyrosine residues in vascular endothelial growth factor receptor-2/FLK-1 involved in activation of phosphatidylinositol 3-kinase and cell proliferation. J. Biol. Chem.276:17686-17692. [[PubMed]
- 8. Fanto, M., and MMlodzik. 1999. Asymmetric Notch activation specifies photoreceptors R3 and R4 and planar polarity in the Drosophila eye. Nature397:523-526. [[PubMed][Google Scholar]
- 9. Fong, G. H., J. Rossant, M. Gertsenstein, and M. L. Breitman. 1995. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature376:66-70. [[PubMed]
- 10. Hanahan, D. 1997. Signaling vascular morphogenesis and maintenance. Science277:48-50. [[PubMed]
- 11. He, T. C., S. Zhou, L. T. da Costa, J. Yu, K. W. Kinzler, and B. Vogelstein. 1998. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. USA95:2509-2514.
- 12. Henderson, A. M., S. J. Wang, A. C. Taylor, M. Aitkenhead, and C. C. Hughes. 2001. The basic helix-loop-helix transcription factor HESR1 regulates endothelial cell tube formation. J. Biol. Chem.276:6169-6176. [[PubMed]
- 13. Hrabe de Angelis, M., J. McIntyre second, and A. Gossler. 1997. Maintenance of somite borders in mice requires the Delta homologue Dll1. Nature386:717-721. [[PubMed]
- 14. Huang, K., C. Andersson, G. M. Roomans, N. Ito, and L. Claesson-Welsh. 2001. Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers. Int. J. Biochem. Cell Biol.33:315-324. [[PubMed]
- 15. Huppert, S. S., A. Le, E. H. Schroeter, J. S. Mumm, M. T. Saxena, L. A. Milner, and R. Kopan. 2000. Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1. Nature405:966-970. [[PubMed]
- 16. Joutel, A., C. Corpechot, A. Ducros, K. Vahedi, H. Chabriat, P. Mouton, S. Alamowitch, V. Domenga, M. Cecillion, E. Marechal, J. Maciazek, C. Vayssiere, C. Cruaud, E. A. Cabanis, M. M. Ruchoux, J. Weissenbach, J. F. Bach, M. G. Bousser, and E. Tournier-Lasserve. 1996. Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia. Nature383:707-710. [[PubMed]
- 17. Kaipainen, A., J. Korhonen, T. Mustonen, V. W. van Hinsbergh, G. H. Fang, D. Dumont, M. Breitman, and K. Alitalo. 1995. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc. Natl. Acad. Sci. USA92:3566-3570.
- 18. Kitamura, T., Y. Kitamura, S. Kuroda, Y. Hino, M. Ando, K. Kotani, H. Konishi, H. Matsuzaki, U. Kikkawa, W. Ogawa, and M. Kasuga. 1999. Insulin-induced phosphorylation and activation of cyclic nucleotide phosphodiesterase 3B by the serine-threonine kinase Akt. Mol. Cell. Biol.19:6286-6296.
- 19. Kotani, K., W. Ogawa, Y. Hino, T. Kitamura, H. Ueno, W. Sano, C. Sutherland, D. K. Granner, and M. Kasuga. 1999. Dominant negative forms of Akt (protein kinase B) and atypical protein kinase Cλ do not prevent insulin inhibition of phosphoenolpyruvate carboxykinase gene transcription. J. Biol. Chem.274:21305-21312. [[PubMed]
- 20. Krebs, L. T., Y. Xue, C. R. Norton, J. R. Shutter, M. Maguire, J. P. Sundberg, D. Gallahan, V. Closson, J. Kitajewski, R. Callahan, G. H. Smith, K. L. Stark, and T. Gridley. 2000. Notch signaling is essential for vascular morphogenesis in mice. Genes Dev.14:1343-1352.
- 21. Kurooka, H., K. Kuroda, and T. Honjo. 1998. Roles of the ankyrin repeats and C-terminal region of the mouse notch1 intracellular region. Nucleic Acids Res.26:5448-5455.
- 22. Leimeister, C., N. Schumacher, C. Steidl, and M. Gessler. 2000. Analysis of HeyL expression in wild-type and Notch pathway mutant mouse embryos. Mech. Dev.98:175-178. [[PubMed]
- 23. Liu, Z.-J., T. Ueda, T. Miyazaki, N. Tanaka, S. Mine, Y. Tanaka, T. Taniguchi, H. Yamamura, and Y. Minami. 1998. A critical role for cyclin C in promotion of the hematopoietic cell cycle by cooperation with c-Myc. Mol. Cell. Biol.18:3445-3454.
- 24. Liu, Z.-J., H. Haleem-Smith, H. Chen, and H. Metzger. 2001. Unexpected signals in a system subject to kinetic proofreading. Proc. Natl. Acad. Sci. USA98:7289-7294.
- 25. Luo, B., J. C. Aster, R. P. Hasserjian, F. Kuo, and J. Sklar. 1997. Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor. Mol. Cell. Biol.17:6057-6067.
- 26. Mignatti, P., T. Morimoto, and D. B. Rifkin. 1992. Basic fibroblast growth factor, a protein devoid of secretory signal sequence, is released by cells via a pathway independent of the endoplasmic reticulum-Golgi complex. J. Cell Physiol.151:81-93. [[PubMed]
- 27. Missero, C. E., Calautti, R. Eckner, J. Chin, L. H. Tsai, D. M. Livingston, and G. P. Dotto. 1995. Involvement of the cell-cycle inhibitor Cip1/WAF1 and the E1A-associated p300 protein in terminal differentiation. Proc. Natl. Acad. Sci. USA92:5451-5455.
- 28. Mumm, J. S., and R. Kopan. 2000. Notch signaling: from the outside in. Dev. Biol.228:151-165. [[PubMed]
- 29. Nesbit, M., H. K. Nesbit, J. Bennett, T. Andl, M. Y. Hsu, E. Dejesus, M. McBrian, A. R. Gupta, S. L. Eck, and M. Herlyn. 1999. Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes. Oncogene18:6469-6476. [[PubMed]
- 30. Ohtsuka, T., M. Ishibashi, G. Gradwohl, S. Nakanishi, F. Guillemot, and R. Kageyama. 1999. Hes1 and Hes5 as Notch effectors in mammalian neuronal differentiation. EMBO J.18:2196-2207.
- 31. Petrova, T. V., T. Makinen, and K. Alitalo. 1999. Signaling via vascular Endothelial growth factor receptors. Exp. Cell Res.253:117-130. [[PubMed]
- 32. Rangarajan, A., C. Talora, R. Okuyama, M. Nicolas, C. Mammucari, H. Oh, J. C. Aster, S. Krishna, D. Metzger, P. Chambon, L. Miele, M. Aguet, F. Radtke, and G. P. Dotto. 2001. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. EMBO J.20:3427-3436.
- 33. Sakaue, H., W. Ogawa, M. Takata, S. Kuroda, K. Kotani, M. Matsumoto, M. Sakaue, S. Nishio, H. Ueno, and M. Kasuga. 1997. Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3-L1 adipocytes. Mol. Endocrinol.11:1552-1562. [[PubMed]
- 34. Shalaby, F., J. Rossant, T. P. Yamaguchi, M. Gertsenstein, X. F. Wu, M. L. Breitman, and A. C. Schuh. 1995. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature376:62-66. [[PubMed]
- 35. Shutter, J. R., S. Scully, W. Fan, W. G. Richards, J. Kitajewski, G. A. Deblandre, C. R. Kintner, and K. L. Stark. 2000. Dll4, a novel Notch ligand expressed in arterial endothelium. Genes Dev.14:1313-1318.
- 36. Smithers, L., C. Haddon, Y. Jiang, and J. Lewis. 2000. Sequence and embryonic expression of deltaC in the zebrafish. Mech. Dev.90:119-123. [[PubMed]
- 37. Soker, S., S. Takashima, H. Q. Miao, G. Neufeld, and M. Klagsbrun. 1998. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor. Cell92:735-745. [[PubMed]
- 38. Sriuranpong, V., M. W. Borges, R. K. Ravi, D. R. Arnold, B. D. Nelkin, S. B. Baylin, and D. W. Ball. 2001. Notch signaling induces cell cycle arrest in small cell lung cancer cells. Cancer Res.61:3200-3205. [[PubMed]
- 39. Stokoe, D., L. R. Stephens, T. Copeland, P. R. Gaffney, C. B. Reese, G. F. Painter, A. B. Holmes, F. McCormick, and P. T. Hawkins. 1997. Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. Science277:567-570. [[PubMed]
- 40. Thakker, G. D., D. P. Hajjar, W. A. Muller, and T. K. Rosengart. 1999. The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling. J. Biol. Chem.274:10002-10007 [[PubMed]
- 41. Tsai, S., J. Fero, and S. Bartelmez. 2000. Mouse Jagged2 is differentially expressed in hematopoietic progenitors and endothelial cells and promotes the survival and proliferation of hematopoietic progenitors by direct cell-to-cell contact. Blood96:950-957. [[PubMed]
- 42. Uyttendaele, H., G. Marazzi, G. Wu, Q. Yan, D. Sassoon, and J. Kitajewski. 1996. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development122:2251-2259. [[PubMed]
- 43. Uyttendaele, H., J. Ho, J. Rossant, and J. Kitajewski. 2001. Vascular patterning defects associated with expression of activated Notch4 in embryonic endothelium. Proc. Natl. Acad. Sci. USA98:5643-5648.
- 44. Veikkola, T., and KAlitalo. 1999. VEGFs, receptors and angiogenesis. Semin. Cancer Biol.9:211-220. [[PubMed][Google Scholar]
- 45. Velazquez, O. C., R. Snyder, Z.-J. Liu, R. M. Fairman, and M. Herlyn. 2002. Fibroblast-dependent differentiation of human microvascular endothelial cells into capillary-like, three-dimensional networks. FASEB J.16:1316-1318. [[PubMed]
- 46. Villa, N., L. Walker, C. E. Lindsell, J. Gasson, M. L. Iruela-Arispe, and G. Weinmaster. 2001. Vascular expression of Notch pathway receptors and ligands is restricted to arterial vessels. Mech. Dev.108:161-164. [[PubMed]
- 47. Wang, H. U., Z. F. Chen, and D. J. Anderson. 1998. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell93:741-753. [[PubMed]
- 48. Weinmaster, G. 2000. Notch signal transduction: a real rip and more. Curr. Opin. Genet. Dev.10:363-369. [[PubMed]
- 49. Xue, Y., X. Gao, C. E. Lindsell, C. R. Norton, B. Chang, C. Hicks, M. Gendron-Maguire, E. B. Rand, G. Weinmaster, and T. Gridley. 1999. Embryonic lethality and vascular defects in mice lacking the Notch ligand Jagged1. Hum. Mol. Genet.8:723-730. [[PubMed]
- 50. Yancopoulos, G. D., S. Davis, N. W. Gale, J. S. Rudge, S. J. Wiegand, and J. Holash. 2000. Vascular-specific growth factors and blood vessel formation. Nature407:242-248. [[PubMed]
- 51. Zhong, T. P., M. Rosenberg, M. A., Mohideen, B. Weinstein, and M. C. Fishman. 2000. gridlock, an HLH gene required for assembly of the aorta in zebrafish. Science287:1820-1824. [[PubMed]
- 52. Zhong, T. P., S. Childs, J. P. Leu, and M. C. Fishman. 2001. Gridlock signalling pathway fashions the first embryonic artery. Nature414:216-220. [[PubMed]
- 53. Zimrin, A. B., M. S. Pepper, G. A. McMahon, F. Nguyen, R. Montesano, and T. Maciag. 1996. An antisense oligonucleotide to the notch ligand jagged enhances fibroblast growth factor-induced angiogenesis in vitro. J. Biol. Chem.271:32499-32502. [[PubMed]