(Means ± standard deviations

[SD] [n = 3]). ††, P < 0.01 versus control + TNF-α (−); **, P < 0.01 versus none + TNF-α (+). TNF-α augments invasion of P. gingivalis through NF-κB and MAPK pathways To determine whether mRNA synthesis and protein synthesis were required for P. gingivalis invasion, Ca9-22 cells were preincubated with 1 μg/ml of the RNA polymerase II inhibitor actinomycin selleck products D or the protein synthesis inhibitor cycloheximide for 1 h and were then incubated with TNF-α prior to addition of P. gingivalis. Actinomycin D and cycloheximide exhibited significant inhibitory effects on the invasion of P.gingivalis into Ca9-22 cells (Figure 3). The PI3K/Akt signaling pathway is commonly initiated by transmembrane receptor signaling and controls cellular phagocytic responses through multiple downstream targets LOXO-101 concentration that regulate actin polymerization and cytoskeletal arrangements at the target site [34]. In addition, TNF-α activates the PI3K/AKT signaling pathway [35]. Therefore, we examined the relationship between PI3K activity and P. gingivalis invasion in Ca9-22cells. Ca9-22 cells were preincubated with wortmannin at 37°C for 3 h and were then incubated with TNF-α. Treatment with

wortmannin also exhibited significant inhibitory activity towards the invasion of P. gingivalis enhanced by TNF-α (Figure 4). selleck Several lines of evidence indicate that cellular effects of TNF-α were elicited through the activation of MAPK and NF-κB pathways. To explore the contribution of MAPK and NF-κB to TNF-α-augmented invasion of P. gingivalis, we examined whether P. gingivalis is able to invade Ca9-22 cells in the presence or absence of MAPK inhibitors and an NF-κB inhibitor. Ca9-22 cells were preincubated with a p38 inhibitor (SB 203580, 5 μM), JNK inhibitor (SP 600125, 1 μM), ERK inhibitor (PD 98059, 5 μM) or NF-κB inhibitor (PDTC, 5 μM) for 1 h and were then incubated with TNF-α prior to addition of others P. gingivalis. SB 203580 and SP 600125 exhibited significant inhibitory effects on the invasion of P. gingivalis into Ca9-22 cells (Figure 5A). In contrast, PD 98059 did not prevent the invasion of P. gingivalis augmented by TNF-α. PDTC also exhibited significant inhibitory

activity towards the invasion of P. gingivalis enhanced by TNF-α (Figure 5B). These results suggest that TNF-α augmented invasion of P. gingivalis is mediated by p38 and JNK pathways and activation of NF-κB. Figure 3 TNF-α augments invasion of P. gingivalis through synthesis of mRNAs and proteins. Actinomycin D and cycloheximide inhibited TNF-α-augmented invasion of P. gingivalis in Ca9-22 cells. Confluent Ca9-22 cells were preincubated with 1 μg/ml actinomycin D (Act D) or cycloheximide (CHX) at 37°C for 1 h and were then incubated with TNF-α for 3 h. The cells were further incubated with P. gingivalis (MOI =100) for 1 h. Viable P. gingivalis in the cells was determined as described in Methods. (Means ± standard deviations [SD] [n = 3]). ††, P < 0.01 versus control + TNF-α (−); **, P < 0.