Vascular Endothelial Growth Factor Enhances the Expression of Urokinase Receptor in Human Endothelial Cells via Protein Kinase C Activation

Summary

Among other proteolytic enzymes, the urokinase-type plasminogen activator (u-PA)/plasmin cascade contributes to cell migration and the formation of capillary-like structures in a fibrinous exudate. The u-PA receptor (u-PAR) focuses proteolytical activity on the cell surface of the endothelial cell and hereby accelerates the pericellular matrix degradation. Vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)-2 enhance u-PA receptor expression in human endothelial cells. In this paper we show that the protein kinase C (PKC) inhibitors Ro31-8220 and GF109203X inhibit VEGF₁₆₅-induced u-PAR antigen expression in human endothelial cells, whereas PKC inhibition had no effect on FGF-2-induced u-PAR antigen enhancement. In addition, inhibition of PKC activity had no effect on VEGF_165-or FGF-2-induced proliferation in human endothelial cells. We conclude that VEGF₁₆₅ induces u-PAR via a PKC-dependent pathway, whereas proliferation is induced via a different pathway probably involving tyrosine phosphorylation of proteins downstream of the VEGF receptors.

Keywords

u-PAR - VEGF - PKC - human endothelial cells

• References

• 1 Eliceiri BP, Cheresh DA. The role of alphav integrins during angiogenesis: insights into potential mechanisms of action and clinical development. J Clin Invest 1999; 103: 1227-30.
• 2 Pepper MS, Sappino AP, Stocklin R, Montesano R, Orci L, Vassalli J-D.. Upregulation of urokinase receptor expression on migrating endothelial cells. J Cell Biol 1993; 122: 673-84.
• 3 Bacharach E, Itin A, Keshet E. In vivo patterns of expression of urokinase and its inhibitor PAI- 1 suggest a concerted role in regulating physiological angiogenesis. Proc Natl Acad Sci USA 1992; 89: 10686-90.
• 4 Moscatelli D, Rifkin DB. Membrane and matrix localization of proteinases: a common theme in tumor cell invasion and angiogenesis. Biochim Biophys Acta 1988; 948: 67-85.
• 5 Min HY, Doyle LV, Vitt CR, Zandonella CL, Stratton-Thomas JR, Shuman MA, Rosenberg S. Urokinase receptor antagonists inhibit angiogenesis and primary tumor growth in syngeneic mice. Cancer Res 1996; 56: 2428-33.
• 6 Koolwijk P, Van Erck MGM, De Vree WJA, Vermeer MA, Weich HA, Hanemaaijer R, Van Hinsbergh VWM. Cooperative effect of TNFalpha, bFGF, and VEGF on the formation of tubular structures of human micro-vascular endothelial cells in a fibrin matrix. Role of urokinase activity. J Cell Biol 1996; 132: 1177-88.
• 7 Pollanen J, Hedman K, Nielsen LS, Dano K, Vaheri A. Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts. J Cell Biol 1988; 106: 87-95.
• 8 Loskutoff DJ. A slice of PAI. J Clin Invest 1993; 92: 2563.
• 9 Ellis V, Behrendt N, Danf K. Plasminogen activation by receptor-bound urokinase - A kinetic study with both cell-associated and isolated receptor. J Biol Chem 1991; 266: 12752-8.
• 10 Nykjaer A, Conese M, Christensen EI, Olson D, Cremona O, Gliemann J, Blasi F. Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes. EMBO J 1997; 16: 2610-20.
• 11 Mandriota SJ, Seghezzi G, Vassalli J-D, Ferrara N, Wasi S, Mazzieri R, Mignatti P, Pepper MS. Vascular endothelial growth factor increases urokinase receptor expression in vascular endothelial cells. J Biol Chem 1995; 270: 9709-16.
• 12 Mignatti P, Mazzieri R, Rifkin DB. Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor. J Cell Biol 1991; 113: 1193-201.
• 13 Gospodarowicz D, Abraham JA, Schilling J. Isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells. Proc Natl Acad Sci USA 1989; 86: 7311-5.
• 14 De Vries C, Escobedo JA, Ueno H, Houck K, Ferrara N, Williams LT. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992; 255: 989-91.
• 15 Terman BI, Dougher-Vermazen M, Carrion ME, Dimitrov D, Armellino DC, Gospodarowicz D, Böhlen P. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor. Biochem Biophys Res Commun 1992; 187: 1579-86.
• 16 Ferris FL, Davis MD, Aiello LM. Treatment of diabetic retinopathy. N Engl J Med 1999; 341: 667-78.
• 17 Harada M, Mitsuyama K, Yoshida H, Sakisaka S, Taniguchi E, Kawaguchi T, Ariyoshi M, Saiki T, Sakamoto M, Nagata K, Sata M, Matsuo K, Tanikawa K. Vascular endothelial growth factor in patients with rheumatoid arthritis. Scand J Rheumatol 1998; 27: 377-80.
• 18 Bhushan M, Mclaughlin B, Weiss JB, Griffiths CE. Levels of endothelial cell stimulating angiogenesis factor and vascular endothelial growth factor are elevated in psoriasis. Br J Dermatol 1999; 141: 1054-60.
• 19 Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int 1999; 56: 794-814.
• 20 Brock TA, Dvorak HF, Senger DR. Tumor-Secreted Vascular Permeability Factor Increases Cytosolic Ca2+ and Vonwillebrand Factor Release in Human Endothelial Cells. Am J Pathol 1991; 138: 213-21.
• 21 Waltenberger J, Claesson-Welsh L, Siegbahn A, Shibuya M, Heldin CH. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor. J Biol Chem 1994; 269: 26988-95.
• 22 Waltenberger J, Mayr U, Pentz S, Hombach V. Functional upregulation of the vascular endothelial growth factor receptor KDR by hypoxia. Circulation 1996; 94: 1647-54.
• 23 Kroll J, Waltenberger J. The vascular endothelial growth factor receptor KDR activates multiple signal transduction pathways in porcine aortic endothelial cells. J Biol Chem 1997; 272: 32521-7.
• 24 Xia P, Aiello LP, Ishii H, Jiang ZY, Park DJ, Robinson GS, Takagi H, Newsome WP, Jirousek MR, King GL. Characterization of vascular endothelial growth factor‘s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clin Invest 1996; 98: 2018-26.
• 25 Wu LW, Mayo LD, Dunbar JD, Kessler KM, Baerwald MR, Jaffe EA, Wang D, Warren RS, Donner DB. Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation. J Biol Chem 2000; 275: 5096-103.
• 26 Kroon ME, Koolwijk P, Goor van H, Weidle U, Collen A, Pluijm van der G, Van Hinsbergh VWM. Role and localization of urokinase receptor in the formation of new microvascular structures in fibrin matrices. Am J Pathol 1999; 154: 1731-42.
• 27 Bohuslav J, Horejsi V, Hansmann C, Stöckl J, Weidle UH, Majdic O, Bartke I, Knapp W, Stockinger H. Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes. J Exp Med 1995; 181: 1381-90.
• 28 Van Hinsbergh VWM, Binnema D, Scheffer MA, Sprengers ED, Kooistra T, Rijken DC. Production of plasminogen activators and inhibitor by serially propagated endothelial cells from adult human blood vessels. Arteriosclerosis 1987; 7: 389-400.
• 29 Van Hinsbergh VWM, Sprengers ED, Kooistra T. Effect of thrombin on the production of plasminogen activators and PA inhibitor-1 by human foreskin microvascular endothelial cells. Thromb Haemost 1987; 57: 148-53.
• 30 Defilippi P, Van Hinsbergh VWM, Bertolotto A, Rossino P, Silengo L, Tarone G. Differential distribution and modulation of expression of alpha 1/ beta 1 integrin on human endothelial cells. J Cell Biol 1991; 114: 855-63.
• 31 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal Biochem 1987; 162: 156-9.
• 32 Lansink M, Koolwijk P, Van Hinsbergh VWM, Kooistra T. Effect of steroid hormones and retinoids on the formation of capillary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression. Blood 1998; 92: 927-38.
• 33 Cubellis MV, Wun TC, Blasi F. Receptor-mediated internalization and degradation of urokinase is caused by its specific inhibitor Pai-1. EMBO J 1990; 9: 1079-85.
• 34 Levin EG, Marotti KR, Santell L. Protein kinase-C and the stimulation of tissue plasminogen activator release from human endothelial cells – dependence on the elevation of messenger RNA. J Biol Chem 1989; 264: 16030-6.
• 35 Pepper MS, Ferrara N, Orci L, Montesano R. Potent synergism between vascular endothelial growth factor and basic fibroblast growth factor in the induction of angiogenesis invitro. Biochem Biophys Res Commun 1992; 189: 824-31.
• 36 Jussila L, Valtola R, Partanen TA, Salven P, Heikkila P, Matikainen MT, Renkonen R, Kaipainen A, Detmar M, Tschachler E, Alitalo R, Alitalo K. Lymphatic endothelium and Kaposi‘s sarcoma spindle cells detected by antibodies against the vascular endothelial growth factor receptor-3. Cancer Res 1998; 58: 1599-604.
• 37 Kaipainen A, Korhonen J, Mustonen T, Van Hinsbergh VWM, Fang GH, Dumont D, Breitman M, Alitalo K. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA 1995; 92: 3566-70.
• 38 Barleon B, Hauser S, Schollmann C, Weindel K, MarmÈ D, Yayon A, Weich HA. Differential expression of the two VEGF receptors Flt and Kdr in placenta and vascular endothelial cells. J Cell Biochem 1994; 54: 56-66.
• 39 Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marmé D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood 1996; 87: 3336-43.
• 40 Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase c-dependent, but ras- independent raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells. Oncogene 1999; 18: 2221-30.
• 41 Doanes AM, Hegland DD, Sethi R, Kovesdi I, Bruder JT, Finkel T. VEGF stimulates MAPK through a pathway that is unique for receptor tyrosine kinases. Biochem Biophys Res Commun 1999; 255: 545-8.
• 42 Shen BQ, Lee DY, Zioncheck TF. Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway. J Biol Chem 1999; 274: 33057-63.
• 43 Koyama S, Takagi H, Otani A, Suzuma K, Nishimura K, Honda Y. Tranilast inhibits protein kinase C-dependent signalling pathway linked to angiogenic activities and gene expression of retinal microcapillary endothelial cells. Br J Pharmacol 1999; 127: 537-45.
• 44 Hood J, Granger HJ. Protein kinase G mediates vascular endothelial growth factor-induced Raf-1 activation and proliferation in human endothelial cells. J Biol Chem 1998; 273: 23504-8.
• 45 Yoshiji H, Kuriyama S, Ways DK, Yoshii J, Miyamoto Y, Kawata M, Ikenaka Y, Tsujinoue H, Nakatani T, Shibuya M, Fukui H. Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res 1999; 59: 4413-8.
• 46 Ozaki H, Seo MS, Ozaki K, Yamada H, Yamada E, Okamoto N, Hofmann F, Wood JM, Campochiaro PA. Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am J Pathol 2000; 156: 697-707.
• 47 Klint P, Kanda S, Kloog Y, Claesson-Welsh L. Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation. Oncogene 1999; 18: 3354-64.
• 48 Li H, Lu H, Griscelli F, Opolon P, Sun LQ, Ragot T, Legrand Y, Belin D, Soria J, Soria C, Perricaudet M, Yeh P. Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice. Gene Therapy 1998; 5: 1105-13.