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.


CONCLUSIONS
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.
Xu
Hanshi
H
Department of Rheumatology, First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan Road 2, Guangzhou, Guangdong 510080, Peoples Republic of China. xuhanshi@hotmail.com
Liu
Peng
P
Liang
Liuqin
L
Danesh
Farhad R
FR
Yang
Xiuyan
X
Ye
Yijin
Y
Zhan
Zhongping
Z
Yu
Xueqing
X
Peng
Hui
H
Sun
Lin
L
D000368:Aged; D001172:Arthritis, Rheumatoid / Q000188:drug therapy* / Q000276:immunology / Q000378:metabolism; D002478:Cells, Cultured; D005260:Female; D005799:Genes, Dominant; D017930:Genes, Reporter; D006801:Humans; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology*; D053583:Interleukin-1beta / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D008875:Middle Aged; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000276:immunology; D019821:Simvastatin / Q000494:pharmacology*; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D053583:Interleukin-1beta; D015850:Interleukin-6; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D019821:Simvastatin; D020742:rhoA GTP-Binding Protein