Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha

Free Radic Biol Med. 2003 Nov 1;35(9):1037-50. doi: 10.1016/s0891-5849(03)00459-3.

Abstract

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-kappaB in transformed brain cell lines [C(6) glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-gamma-mediated iNOS mRNA and increased nitric oxide (NO) production. The addition of mevalonate and geranylgeranylpyrophosphate (GGPP) reversed the lovastatin-mediated effect, whereas FPP had no effect. An inhibitor of geranylgeranyltransferase inhibitor (GGTI 298) further induced the cytokine and lovastatin-mediated iNOS expression, suggesting the involvement of geranylgeranylated proteins in the regulation of iNOS. Bacterial toxin B (inactivates RhoA, B, and C; CDC42; Rac proteins), C3 ADP-ribosyltransferase (C3) toxin from C. botulinum (inactivates RhoA, B, and C proteins), and Y-27632 (selective inhibitor of Rho-associated kinases) increased the LPS/IFN-gamma-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-kappaB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKalpha. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-kappaB-luciferase reporter constructs further support these observations. Taken together, these studies show that downregulation of RhoA by lovastatin resulted in increased iNOS expression via the activation of NF-kappaB-CBP/p300 pathway in transformed brain cells.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Brain / cytology
  • Brain / drug effects*
  • Brain / metabolism*
  • Brain / pathology
  • Cell Line, Transformed
  • Cell Line, Tumor
  • Cytokines / metabolism*
  • Enzyme Inhibitors / pharmacology
  • Gene Expression Regulation / drug effects*
  • Humans
  • I-kappa B Kinase
  • Interferon-gamma / metabolism
  • Lipopolysaccharides / pharmacology
  • Lovastatin / pharmacology
  • Mevalonic Acid / pharmacology
  • NF-kappa B / metabolism
  • Nitric Oxide / metabolism
  • Nitric Oxide Synthase / genetics*
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase Type II
  • Protein Prenylation / drug effects
  • Protein-Serine-Threonine Kinases / metabolism*
  • Rats
  • rhoA GTP-Binding Protein / antagonists & inhibitors
  • rhoA GTP-Binding Protein / metabolism*

Substances

  • Cytokines
  • Enzyme Inhibitors
  • Lipopolysaccharides
  • NF-kappa B
  • Nitric Oxide
  • Interferon-gamma
  • Lovastatin
  • NOS2 protein, human
  • Nitric Oxide Synthase
  • Nitric Oxide Synthase Type II
  • Nos2 protein, rat
  • Protein-Serine-Threonine Kinases
  • CHUK protein, human
  • I-kappa B Kinase
  • IKBKB protein, human
  • IKBKE protein, human
  • rhoA GTP-Binding Protein
  • Mevalonic Acid