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

The present study describes the role of RhoA as a negative regulator of iNOS expression via the inactivation of NF-κB in transformed brain cell lines [C6 glioma, human astrocytoma (T98G, A172), neuroblastoma (NEB), and immortal rat astrocytes]. Treatment with lovastatin resulted in the induction of LPS/IFN-γ-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-γ-mediated iNOS expression. Lovastatin treatment induced NO by increasing NF-κB translocation and its association with the CREB-binding protein (CBP/p300) via the downregulation of RhoA. Inhibition of RhoA resulted in increased activation of IKKα. Cotransfection studies with dominant-negative form of RhoA and iNOS-luciferase or NF-κB-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-κB-CBP/p300 pathway in transformed brain cells.