RhoA-mediated, tumor necrosis factor α-induced activation of NF-κB in rheumatoid synoviocytes: Inhibitory effect of simvastatin

Objective. Increasing evidence indicates that RhoA may play a central role in the inflammatory response. This study was conducted to examine the role of RhoA in mediating the activation of NF-κB in tumor necrosis factor α (TNFα)-stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFα-induced activation of RhoA and NF-κB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes. Methods. Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFα and incubated with simvastatin (SMV) (1 μM). RhoA activity was assessed by a pull-down assay. NF-κB DNA binding activity and nuclear translocation of NF-κB were measured by a sensitive multi-well colorimetric assay and confocal fluorescence microscopy, respectively. Results. TNFα stimulation elicited a robust increase in RhoA activity in a dose-dependent manner, and SMV mitigated this increase. TNFα also hastened NF-κB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IκB, and secretion of interleukin-1β (IL-1β) and IL-6. SMV prevented the increase in NF-κB activation and rise in IL-1β and IL-6 levels induced by TNFα, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF-κB and RhoA. Furthermore, cotransfection with a dominant-negative mutant of RhoA demonstrated that the TNFα-induced signaling pathway involved sequential activation of RhoA, leading to NF-κB activation and, ultimately, to secretion of cytokines. Conclusion. This study identifies RhoA as the key regulator of TNFα-induced NF-κB activation, which ultimately results in the secretion of proinflammatory cytokines in rheumatoid synoviocytes. The findings provide a new rationale for the antiinflammatory effects of statins in inflammatory arthritis.