Figure 6

CCN4 induced NF-κB activation through αvβ5 integrin/PI3K/Akt pathway. (A-C) OASFs were incubated with various concentrations of CCN4 or pretreated with LY294002, Wortmannin or Akti, PDTC or TPCK for 30 minutes or transfected with PI3K, Akt, IKKα, and IKKβ mutant before exposure to CCN4. NF-κB luciferase activity was measured, and the results were normalized to the β-galactosidase activity. (D) OASFs were pretreated with LY294002, Wortmannin or Akti for 30 minutes followed by stimulation with CCN4 for 60 minutes, and p-p65 expression was examined by Western blot analysis. (E) OASFs were pretreated with LY294002, Wortmannin or Akti for 30 minutes then stimulated with CCN4 for 120 minutes, and p65 immunofluorescence staining was examined. (F) OASFs were pretreated with PDTC and TPCK for 30 minutes followed by stimulation with CCN4 for 30 minutes, and p-Akt expression was examined by Western blot analysis. Results are expressed as the mean ± SEM (n = 4). *, P <0.05 compared with control; #, P <0.05 compared with the CCN4-treated group. OASFs, osteoarthritis synovial fibroblasts; PDTC, pyrrolidine-dithiocarbamate; SEM, standard error of the mean, TPCK, L-1-tosylamido-2-phenylenylethyl chloromethyl ketone.