gingivalis infected ARRY-438162 osteoblasts at any of the experimental time points (data not shown). Figure 2 Actin filament rearrangement is essential for P. gingivalis invasion of osteoblasts. A. Osteoblast nuclei, actin and P. gingivalis are indicated by blue, red or green fluorescence, respectively. No appreciable change in actin filament organization was seen 30 min after infection. At 3 h, actin relocated to the periphery of the osteoblasts, leaving a void space surrounding the osteoblast nuclei occupied by P. gingivalis. Twenty-four hours after infection, actin became more condensed and formed a cortical outer shell. The number of perinuclear P. gingivalis was also significantly increased.
Addition of the actin disrupting agent, cytochalasin D, reduced the number of osteoblasts with P. gingivalis invasion. Notice that actin had now become Selleck SB202190 disorganized, as demonstrated by the punctuated see more pattern. B. Quantitative analysis of confocal images demonstrated that P. gingivalis invasion of osteoblasts was inhibited by
the disruption of actin filaments. Abbreviations: min, minute; h, hour; Ctrl and CT, control, non-infected osteoblasts; PG, P. gingivalis. Scale bar = 20 μm. * denotes P < 0.05. To investigate whether actin rearrangement is necessary for P. gingivalis entry into osteoblasts, the actin-disrupting agent cytochalasin D was added to the cultures together with the bacteria. Figure 2A shows that cytochalasin D treated osteoblasts demonstrated disorganized and punctuated actin filaments. Quantitative image analysis demonstrated that the bacterial invasion of osteoblasts was significantly less following treatment with cytochalasin D compared with untreated cells (Figure 2B), indicating that actin rearrangement is essential
for P. gingivalis invasion of osteoblasts. The JNK pathway is activated in osteoblasts upon repeated infection with P. gingivalis Because the MAPK pathway is activated by many host-pathogen interactions, we investigated whether this pathway is activated in osteoblasts infected with P. gingivalis. Considering that periodontitis is a chronic infectious disease, Ribonucleotide reductase we inoculated P. gingivalis into osteoblast cultures repeatedly every other day for up to 3 weeks to mimic the chronic nature of this disease. Western blot analysis showed that phosphorylated JNK (p-JNK) bands were more intense in treated cells than in control cells from day 7 to day 21 (Figure 3A), whereas there was no noticeable change in ERK and p38 (data not shown). After normalization to actin, quantitative densitometric analysis showed that the p-JNK/JNK ratio was significantly higher in the infected osteoblasts compared with control cells (Figure 3B), indicating that the JNK pathway was activated in osteoblasts chronically infected with P. gingivalis. Figure 3 JNK pathway is activated in osteoblasts upon repeated P. gingivalis infection. A. P.