RhoA-mediated, tumor necrosis factor alpha-induced activation of NF-kappaB 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-kappaB in tumor necrosis factor alpha (TNFalpha)-stimulated rheumatoid synoviocytes, and to evaluate the modulatory effects of statins on the TNFalpha-induced activation of RhoA and NF-kappaB and the secretion of proinflammatory cytokines by rheumatoid synoviocytes.

METHODS: Rheumatoid synoviocytes obtained from patients with active rheumatoid arthritis were stimulated with TNFalpha and incubated with simvastatin (SMV) (1 muM). RhoA activity was assessed by a pull-down assay. NF-kappaB DNA binding activity and nuclear translocation of NF-kappaB were measured by a sensitive multiwell colorimetric assay and confocal fluorescence microscopy, respectively.

RESULTS: TNFalpha stimulation elicited a robust increase in RhoA activity in a dose-dependent manner, and SMV mitigated this increase. TNFalpha also hastened NF-kappaB nuclear translocation of subunit p65 and increased DNA binding activity, luciferase reporter gene expression, degradation of IkappaB, and secretion of interleukin-1beta (IL-1beta) and IL-6. SMV prevented the increase in NF-kappaB activation and rise in IL-1beta and IL-6 levels induced by TNFalpha, whereas mevalonate and geranylgeranyl pyrophosphate reversed the inhibitory effects of SMV on activation of NF-kappaB and RhoA. Furthermore, cotransfection with a dominant-negative mutant of RhoA demonstrated that the TNFalpha-induced signaling pathway involved sequential activation of RhoA, leading to NF-kappaB activation and, ultimately, to secretion of cytokines.

CONCLUSION: This study identifies RhoA as the key regulator of TNFalpha-induced NF-kappaB 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.