Figure 6
PKR is the primary molecule exhibiting DPIT activity. (a) Microvesicles (MVs) purified from DP-1 sup possessed the ability to induce TNF-α secretion in dTHP-1 cells, and treatment of DP-1 cells with PKR inhibitor (C-16) reduced the TNF-α-inducible ability of these MVs. DP-1 cells were treated with C-16 (0, 0.5, or 1 μM) for 24 hrs and then MVs were purified from the supernatants. Then, dTHP-1 cells were stimulated with the various concentrations of these purified MVs for 2 hrs and the amounts of secreted TNF-α were quantified. (b) Pre-treatment of dTHP-1 cells with a PKR inhibitor (C-16: 1 μM, 2-AP: 20 mM) also suppressed TNF-α production after stimulation with DP-1 sup and PriDPC-1 sup. Note that pre-treatment with inhibitors of NFκB (BAY 11-70: 10 μM; Parthenolide: 1 μM), JNK (SP600125: 10 μM), p-38 SB203580: 10 μM), and ERK (FR180204: 10 μM) also suppressed TNF-α production, while pre-treatment with inhibitors of RIPK1 (necrtostatin-1: 10 μM) and CamKII (KN-93: 10 μM) did not alter TNF-α production. (c) In contrast, treatment of dTHP-1 cells with siRNAs to successfully down-regulate endogenous PKR (left) did not influence TNF-α production after stimulation with supernatants from DP-1 (right). (d) Culture supernatants from dental pulp cells (DP-1, priDPC-1), but not those from gingival fibroblasts, markedly activated JNK, p38, and NF-κB in dTHP-1 cells. The ability of DP-1 sup and PriDPC-1 sup in cell activation is extremely powerful as compared with that of LPS (right). n.s.: not significant. Data represent means ± SD of at least two independent experiments with identical results. Statistical analyses were performed by one-way ANOVA, followed by Tukey’s test (a,c) or Dunnett’s test (b). ***p < 0.001, significant differences versus dTHP-1 cells treated with equivalent amounts of MVs from non-treated dDP-1 cells (a) or dTHP-1 cells treated with DMSO (b).
