title: Involvement of nitric oxide synthase in matrix metalloproteinase-9- and/or urokinase plasminogen activator receptor-mediated glioma cell migration.
abstract: BACKGROUND
  Src tyrosine kinase activates inducible nitric oxide synthase (iNOS) and, in turn, nitric oxide production as a means to transduce cell migration. Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Our recent studies have clearly demonstrated the role of α9β1 integrin in matrix metalloproteinase-9 (MMP-9) and/or urokinase plasminogen activator receptor (uPAR)-mediated glioma cell migration. In the present study, we evaluated the involvement of α9β1 integrin-iNOS pathway in MMP-9- and/or uPAR-mediated glioma cell migration.
  
  
  METHODS
  MMP-9 and uPAR shRNAs and overexpressing plasmids were used to downregulate and upregulate these molecules, respectively in U251 glioma cells and 5310 glioma xenograft cells. The effect of treatments on migration and invasion potential of these glioma cells were assessed by spheroid migration, wound healing, and Matrigel invasion assays. In order to attain the other objectives we also performed immunocytochemical, immunohistochemical, RT-PCR, Western blot and fluorescence-activated cell sorting (FACS) analysis.
  
  
  RESULTS
  Immunohistochemical analysis revealed the prominent association of iNOS with glioblastoma multiforme (GBM). Immunofluorescence analysis showed prominent expression of iNOS in glioma cells. MMP-9 and/or uPAR knockdown by respective shRNAs reduced iNOS expression in these glioma cells. RT-PCR analysis revealed elevated iNOS mRNA expression in either MMP-9 or uPAR overexpressed glioma cells. The migration potential of MMP-9- and/or uPAR-overexpressed U251 glioma cells was significantly inhibited after treatment with L-NAME, an inhibitor of iNOS. Similarly, a significant inhibition of the invasion potential of the control or MMP-9/uPAR-overexpressed glioma cells was noticed after L-NAME treatment. A prominent reduction of iNOS expression was observed in the tumor regions of nude mice brains, which were injected with 5310 glioma cells, after MMP-9 and/or uPAR knockdown. Protein expressions of cSrc, phosphoSrc and p130Cas were reduced with simultaneous knockdown of both MMP-9 and uPAR.
  
  
  CONCLUSIONS
  Taken together, our results from the present and earlier studies clearly demonstrate that α9β1 integrin-mediated cell migration utilizes the iNOS pathway, and inhibition of the migratory potential of glioma cells by simultaneous knockdown of MMP-9 and uPAR could be attributed to the reduced α9β1 integrin and iNOS levels.
authors: Thompson
authors: T
authors: Zhuang
authors: Bharath
authors: B
authors: Chelluboina
authors: Shivani
authors: S
authors: Ponnala
authors: Kiran Kumar
authors: KK
authors: Velpula
authors: Azeem A
authors: AA
authors: Rehman
authors: Chandramu
authors: C
authors: Chetty
authors: Eleonora
authors: E
authors: Zakharian
authors: Jasti S
authors: JS
authors: Rao
authors: Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, One Illini Drive, Peoria, IL 61605, USA. krishnav@uic.edu.
authors: Krishna Kumar
authors: KK
authors: Veeravalli
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D004195:Disease Models, Animal; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005910:Glioma / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D064593:Heterografts; D006801:Humans; D016023:Integrins / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D055293:Receptors, Urokinase Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D016023:Integrins; D055293:Receptors, Urokinase Plasminogen Activator; C113256:integrin alpha 9 beta 1; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D020780:Matrix Metalloproteinase 9; D019331:NG-Nitroarginine Methyl Ester