Rho A negatively regulates cytokine-mediated inducible nitric oxide synthase expression in brain-derived transformed cell lines: negative regulation of IKKalpha.
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.
Department of Pediatrics, Medical University of South Carolina, Charleston, SC 29425, USA.
Ramandeep
R
Rattan
Shailendra
S
Giri
Avtar K
AK
Singh
Inderjit
I
Singh
D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D016207:Cytokines / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D051550:I-kappa B Kinase; D007371:Interferon-gamma / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008148:Lovastatin / Q000494:pharmacology; D008798:Mevalonic Acid / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D017368:Protein Prenylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*
D016207:Cytokines; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009569:Nitric Oxide; D007371:Interferon-gamma; D008148:Lovastatin; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human; D020742:rhoA GTP-Binding Protein; D008798:Mevalonic Acid