Angiopoietin-1 (Ang1) signals via the receptor tyrosine kinase Tie2 which exists in complex with the related protein Tie1 at the endothelial cell surface. Tie1 undergoes regulated ectodomain cleavage in response to phorbol esters, vascular endothelial growth factor (VEGF) and tumour necrosis factor-α (TNFα). Recently phorbol esters and VEGF were found also to stimulate ectodomain cleavage of Tie2. Here we investigate for the first time the effects of factors activating ectodomain cleavage on both Tie1 and Tie2 within the same population of cells, and their impact on angiopoietin signalling. We find that phorbol ester and VEGF activated Tie1 cleavage within minutes followed by restoration to control levels by 24 h. However, several hours of PMA and VEGF treatment were needed to elicit a detectable decrease in cellular Tie2, with complete loss seen at 24 h of PMA treatment. TNFα stimulated Tie1 cleavage, and induced a sustained decrease in cellular Tie1 over 24 h whilst increasing cellular Tie2. These differential effects of agonists on Tie1 and Tie2 result in dynamic modulation of the cellular Tie2:Tie1 ratio. To assess the impact of this on Ang1 signalling cells were stimulated with VEGF and TNFα for differing times and Ang1-induced Tie2 phosphorylation examined. Elevated Tie2:Tie1, in response to acute VEGF treatment or chronic TNFα, was associated with increased Ang1-activated Tie2 in cells. These data demonstrate cellular levels of Tie1 and Tie2 are differentially regulated by pathophysiologically relevant agonists resulting in dynamic control of the cellular Tie2:Tie1 balance and modulation of Ang1 signalling. These findings highlight the importance of regulation of signalling at the level of the receptor. Such control may be an important adaptation to allow modulation of cellular signalling responses in systems in which the activating ligand is normally present in excess or where the ligand provides a constitutive maintenance signal.
References
[1]
Peters KG, Kontos CD, Lin PC, Wong AL, Rao P, et al. (2004) Functional Significance of Tie2 Signaling in the Adult Vasculature. Recent Prog Horm Res 59: 51–71.
[2]
Brindle NPJ, Saharinen P, Alitalo K (2006) Signaling and Functions of Angiopoietin-1 in Vascular Protection. Circ Res 98: 1014–1023.
[3]
Jones PF (2003) Not just angiogenesis–wider roles for the angiopoietins. J Pathol 201: 515–527.
[4]
Suri C, Jones PF, Patan S, Bartunkova S, Maisonpierre PC, et al. (1996) Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87: 1171–1180.
[5]
Gale N, Thurston G, Hackett S, Renard R, Wang Q, et al. (2002) Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by angiopoietin-1. Dev Cell 3: 411.
[6]
Wong AL, Haroon ZA, Werner S, Dewhirst MW, Greenberg CS, et al. (1997) Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues. Circ Res 81: 567–574.
[7]
Maisonpierre PC, Suri C, Jones PF, Bartunkova S, Wiegand SJ, et al. (1997) Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 277: 55–60.
[8]
Davis S, Aldrich TH, Jones PF, Acheson A, Compton DL, et al. (1996) Isolation of angiopoietin-1, a ligand for the TIE2 receptor, by secretion-trap expression cloning. Cell 87: 1161–1169.
[9]
Augustin HG, Young Koh G, Thurston G, Alitalo K (2009) Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol 10: 165–177.
[10]
Marron MB, Hughes DP, Edge MD, Forder CL, Brindle NPJ (2000) Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2. J Biol Chem 275: 39741–39746.
[11]
Partanen J, Armstrong E, Makela TP, Korhonen J, Sandberg M, et al. (1992) A novel endothelial cell surface receptor tyrosine kinase with extracellular epidermal growth factor homology domains. Mol Cell Biol 12: 1698–1707.
[12]
Dumont DJ, Yamaguchi TP, Conlon RA, Rossant J, Breitman ML (1992) tek, a novel tyrosine kinase gene located on mouse chromosome 4, is expressed in endothelial cells and their presumptive precursors. Oncogene 7: 71–80.
[13]
Runting AS, Stacker SA, Wilks AF (1993) tie2, a putative protein tyrosine kinase from a new class of cell surface receptor. Growth Factors 9: 99–105.
[14]
Dumont DJ, Gradwohl GJ, Fong GH, Auerbach R, Breitman ML (1993) The endothelial-specific receptor tyrosine kinase, tek, is a member of a new subfamily of receptors. Oncogene 8: 1293–1301.
[15]
Maisonpierre PC, Goldfarb M, Yancopoulos GD, Gao G (1993) Distinct rat genes with related profiles of expression define a TIE receptor tyrosine kinase family. Oncogene 8: 1631–1637.
[16]
Ziegler S, Bird T, Schneringer J, Schooley K, Baum P (1993) Molecular cloning and characterization of a novel receptor protein tyrosine kinase from human placenta. Oncogene 8: 663–670.
[17]
Marron MB, Singh H, Tahir TA, Kavumkal J, Kim H-Z, et al. (2007) Regulated proteolytic processing of Tie1 modulates ligand responsiveness of the receptor tyrosine kinase Tie2. J Biol Chem 282: 30509–30517.
[18]
Yuan HT, Venkatesha S, Chan B, Deutsch U, Mammoto T, et al. (2007) Activation of the orphan endothelial receptor Tie1 modifies Tie2-mediated intracellular signaling and cell survival. FASEB J 21: 3171–3183.
[19]
Reusch P, Barleon B, Weindel K, Martiny-Baron G, Godde A, et al. (2001) Identification of a soluble form of the angiopoietin receptor TIE-2 released from endothelial cells and present in human blood. Angiogenesis 4: 123–131.
[20]
Findley CM, Cudmore MJ, Ahmed A, Kontos CD (2007) VEGF Induces Tie2 Shedding via a Phosphoinositide 3-Kinase/Akt-Dependent Pathway to Modulate Tie2 Signaling. Arterioscler Thromb Vasc Biol 27: 2619–2626.
[21]
Onimaru M, Yonemitsu Y, Suzuki H, Fujii T, Sueishi K (2010) An Autocrine Linkage Between Matrix Metalloproteinase-14 and Tie-2 Via Ectodomain Shedding Modulates Angiopoietin-1-Dependent Function in Endothelial Cells. Arterioscler Thromb Vasc Biol 30: 818–826.
[22]
Yabkowitz R, Meyer S, Black T, Elliott G, Merewether LA, et al. (1999) Inflammatory cytokines and vascular endothelial growth factor stimulate the release of soluble tie receptor from human endothelial cells via metalloprotease activation. Blood 93: 1969–1979.
[23]
Yabkowitz R, Myer S, Yanagihara D, Brankow D, Staley T, et al. (1997) Regulation of tie receptor expression on human endothelial cells by protein kinase C-mediated release of soluble tie. Blood 90: 706–715.
[24]
McCarthy MJ, Burrows R, Bell SC, Christie G, Bell PRF, et al. (1999) Potential roles of metalloprotease mediated ectodomain cleavage in signaling by the endothelial receptor tyrosine kinase Tie-1. Lab Invest 79: 889–895.
[25]
Chen-Konak L, Guetta-Shubin Y, Yahav H, Shay-Salit A, Zilberman M, et al. (2003) Transcriptional and post-translation regulation of the Tie1 receptor by fluid shear stress changes in vascular endothelial cells. FASEB J 17: 2121–2123.
Willam C, Koehne P, Jurgensen JS, Grafe M, Wagner KD, et al. (2000) Tie2 Receptor Expression Is Stimulated by Hypoxia and Proinflammatory Cytokines in Human Endothelial Cells. Circ Res 87: 370–377.
[28]
Ni CY, Murphy MP, Golde TE, Carpenter G (2001) gamma -Secretase cleavage and nuclear localization of ErbB-4 receptor tyrosine kinase. Science 294: 2179–2181.
[29]
Wilhelmsen K, van der Geer P (2004) Phorbol 12-Myristate 13-Acetate-Induced Release of the Colony-Stimulating Factor 1 Receptor Cytoplasmic Domain into the Cytosol Involves Two Separate Cleavage Events. Mol Cell Biol 24: 454–464.
[30]
Linggi B, Cheng QC, Rao AR, Carpenter G (2006) The ErbB-4 s80 intracellular domain is a constitutively active tyrosine kinase. Oncogene 25: 160–163.
[31]
Cabrera N, Diaz-Rodriguez E, Becker E, Martin-Zanca D, Pandiella A (1996) TrkA receptor ectodomain cleavage generates a tyrosine phosphorylated cell-associated fragment. J Cell Biol 132: 427–436.
[32]
Oh H, Takagi H, Suzuma K, Otani A, Matsumura M, et al. (1999) Hypoxia and vascular endothelial growth factor selectively up-regulate angiopoietin-2 in bovine microvascular endothelial cells. J Biol Chem 274: 15732–15739.
[33]
Mandriota SJ, Pepper MS (1998) Regulation of angiopoietin-2 mRNA levels in bovine microvascular endothelial cells by cytokines and hypoxia. Circ Res 83: 852–859.
[34]
Tadros A, Hughes DP, Dunmore BJ, Brindle NPJ (2003) ABIN-2 protects endothelial cells from death and has a role in the antiapoptotic effect of angiopoietin-1. Blood 102: 4407–4409.
[35]
Singh H, Milner CS, Aguilar Hernandez MM, Patel N, Brindle NP (2009) Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases. Cell Signal 21: 1346–1350.
[36]
Matthews JA, Batki A, Hynds C, Kricka LJ (1985) Enhanced chemiluminescent method for the detection of DNA dot-hybridization assays. Anal Biochem 151: 205–209.
