title: Suppression of uPA and uPAR attenuates angiogenin mediated angiogenesis in endothelial and glioblastoma cell lines.
abstract: BACKGROUND
  In our earlier reports, we showed that downregulation of uPA and uPAR inhibited glioma tumor angiogenesis in SNB19 cells, and intraperitoneal injection of a hairpin shRNA expressing plasmid targeting uPA and uPAR inhibited angiogenesis in nude mice. The exact mechanism by which inhibition of angiogenesis takes place is not clearly understood.
  
  
  RESULTS
  In the present study, we have attempted to investigate the mechanism by which uPA/uPAR downregulation by shRNA inhibits angiogenesis in endothelial and glioblastoma cell lines. uPA/uPAR downregulation by shRNA in U87 MG and U87 SPARC co-cultures with endothelial cells inhibited angiogenesis as assessed by in vitro angiogenesis assay and in vivo dorsal skin-fold chamber model in nude mice. Protein antibody array analysis of co-cultures of U87 and U87 SPARC cells with endothelial cells treated with pU2 (shRNA against uPA and uPAR) showed decreased angiogenin secretion and angiopoietin-1 as well as several other pro-angiogenic molecules. Therefore, we investigated the role of angiogenin and found that nuclear translocation, ribonucleolytic and 45S rRNA synthesis, which are all critical for angiogenic function of angiogenin, were significantly inhibited in endothelial cells transfected with uPA, uPAR and uPA/uPAR when compared with controls. Moreover, uPA and uPAR downregulation significantly inhibited the phosphorylation of Tie-2 receptor and also down regulated FKHR activation in the nucleus of endothelial cells via the GRB2/AKT/BAD pathway. Treatment of endothelial cells with ruPA increased angiogenin secretion and angiogenin expression as determined by ELISA and western blotting in a dose-dependent manner. The amino terminal fragment of uPA down regulated ruPA-induced angiogenin in endothelial cells, thereby suggesting that uPA plays a critical role in positively regulating angiogenin in glioblastoma cells.
  
  
  CONCLUSIONS
  Taken together, our results suggest that uPA/uPAR downregulation suppresses angiogenesis in endothelial cells induced by glioblastoma cell lines partially by downregulation of angiogenin and by inhibition of the angiopoietin-1/AKT/FKHR pathway.
authors.affiliation: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, Illinois, United States of America.
authors.forename: Hari
authors.initials: H
authors.lastname: Raghu
authors.forename: Sajani S
authors.initials: SS
authors.lastname: Lakka
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Gondi
authors.forename: Sanjeeva
authors.initials: S
authors.lastname: Mohanam
authors.forename: Dzung H
authors.initials: DH
authors.lastname: Dinh
authors.forename: Meena
authors.initials: M
authors.lastname: Gujrati
authors.forename: Jasti S
authors.initials: JS
authors.lastname: Rao
mesh_terms: D051696:Activating Transcription Factors / Q000378:metabolism; D021581:Active Transport, Cell Nucleus / Q000235:genetics; D042683:Angiopoietin-1 / Q000378:metabolism; D042702:Angiopoietin-2 / Q000378:metabolism; D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000235:genetics; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005909:Glioblastoma / Q000098:blood supply / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D051379:Mice; D009389:Neovascularization, Pathologic / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000235:genetics; D012335:RNA, Ribosomal / Q000096:biosynthesis; D034741:RNA, Small Interfering / Q000235:genetics; D042787:Receptor, TIE-2 / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator / Q000172:deficiency* / Q000235:genetics*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism* / Q000494:pharmacology; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics; D014568:Urokinase-Type Plasminogen Activator / Q000172:deficiency* / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology
chemical_list: D051696:Activating Transcription Factors; D042683:Angiopoietin-1; D042702:Angiopoietin-2; D012335:RNA, Ribosomal; D034741:RNA, Small Interfering; C088179:RNA, ribosomal, 45S; D055293:Receptors, Urokinase Plasminogen Activator; D042787:Receptor, TIE-2; C046366:angiogenin; D012259:Ribonuclease, Pancreatic; D014568:Urokinase-Type Plasminogen Activator
references.citation: Ann N Y Acad Sci. 1992 Dec 4;667:1-12
references.pmid: 1339240
references.citation: J Biol Chem. 2005 Jun 10;280(23):22445-53
references.pmid: 15781448
references.citation: Eur J Cancer. 1996 Dec;32A(14):2386-93
references.pmid: 9059326
references.citation: J Biol Chem. 2002 Apr 5;277(14):12479-85
references.pmid: 11805108
references.citation: Biochemistry. 2003 Jan 14;42(1):121-8
references.pmid: 12515546
references.citation: Am J Pathol. 1991 May;138(5):1059-67
references.pmid: 1850957
references.citation: J Biol Chem. 2001 Jul 13;276(28):26516-25
references.pmid: 11346644
references.citation: J Urol. 2001 Dec;166(6):2495-9
references.pmid: 11696817
references.citation: J Neurooncol. 2002 Dec;60(3):213-26
references.pmid: 12510773
references.citation: Cancer Sci. 2007 Feb;98(2):253-8
references.pmid: 17233842
references.citation: Oncogene. 2003 Jan 23;22(3):392-400
references.pmid: 12545160
references.citation: Cancer Res. 2004 Jun 15;64(12):4069-77
references.pmid: 15205313
references.citation: Hum Pathol. 2001 Oct;32(10):1064-70
references.pmid: 11679940
references.citation: Biochem Soc Trans. 2003 Feb;31(Pt 1):292-7
references.pmid: 12546704
references.citation: Clin Exp Metastasis. 1999;17(7):617-21
references.pmid: 10845561
references.citation: Proc Natl Acad Sci U S A. 2006 Sep 26;103(39):14519-24
references.pmid: 16971483
references.citation: Int J Oncol. 2009 Mar;34(3):707-15
references.pmid: 19212676
references.citation: Matrix Biol. 2000 Dec;19(7):569-80
references.pmid: 11102747
references.citation: Int J Oncol. 2006 Dec;29(6):1349-57
references.pmid: 17088972
references.citation: Proc Natl Acad Sci U S A. 1987 Dec;84(24):8783-7
references.pmid: 3122207
references.citation: J Biol Chem. 1994 Dec 23;269(51):32380-8
references.pmid: 7528215
references.citation: World J Gastroenterol. 2009 Nov 21;15(43):5432-41
references.pmid: 19916173
references.citation: Arch Immunol Ther Exp (Warsz). 1999;47(5):299-305
references.pmid: 10604235
references.citation: Cancer Sci. 2003 Jan;94(1):43-9
references.pmid: 12708473
references.citation: Science. 1996 Sep 13;273(5281):1551-5
references.pmid: 8703217
references.citation: Nat Rev Cancer. 2003 Jul;3(7):489-501
references.pmid: 12835669
references.citation: Int J Dev Neurosci. 1999 Aug-Oct;17(5-6):463-72
references.pmid: 10571408
references.citation: Clin Cancer Res. 2001 Aug;7(8):2519-26
references.pmid: 11489835
references.citation: Science. 1998 Oct 16;282(5388):468-71
references.pmid: 9774272
references.citation: Oncogene. 2002 Nov 7;21(51):7824-30
references.pmid: 12420219
references.citation: Int J Oncol. 2006 Apr;28(4):831-9
references.pmid: 16525631
references.citation: J Neuropathol Exp Neurol. 1999 Oct;58(10):1029-40
references.pmid: 10515226
references.citation: Annu Rev Microbiol. 1996;50:645-77
references.pmid: 8905094
references.citation: Adv Exp Med Biol. 2000;476:35-46
references.pmid: 10949653
references.citation: Int J Oncol. 2008 Nov;33(5):937-47
references.pmid: 18949356
references.citation: Proc Natl Acad Sci U S A. 1998 Aug 18;95(17):9791-5
references.pmid: 9707554
references.citation: Cancer Res. 2005 Feb 15;65(4):1352-60
references.pmid: 15735021
references.citation: J Clin Invest. 1996 Jan 15;97(2):273-4
references.pmid: 8567944
references.citation: Oncogene. 2007 Jun 14;26(28):4084-94
references.pmid: 17213807
references.citation: FASEB J. 2000 Dec;14(15):2589-600
references.pmid: 11099478
references.citation: FASEB J. 2003 Apr;17(6):752-4
references.pmid: 12594181
references.citation: Lab Invest. 1999 Feb;79(2):213-23
references.pmid: 10068209
references.citation: Oncogene. 2005 Jan 13;24(3):445-56
references.pmid: 15558023
references.citation: Cancer Res. 1994 Sep 15;54(18):5016-20
references.pmid: 8069869
references.citation: Front Biosci. 1999 Feb 15;4:D178-87
references.pmid: 9989951
references.citation: Cancer Res. 1992 Dec 1;52(23):6553-60
references.pmid: 1384965
references.citation: Clin Cancer Res. 1999 May;5(5):1125-30
references.pmid: 10353747
references.citation: Clin Cancer Res. 2007 Jul 15;13(14):4051-60
references.pmid: 17634529
references.citation: Cancer Lett. 2005 Nov 8;229(1):13-23
references.pmid: 16157214
references.citation: J Cell Biol. 2005 Sep 12;170(6):993-1004
references.pmid: 16157706