Porphyromonas gingivalis fimbriae inhibit caspase-3-mediated apoptosis of monocytic THP-1 cells under growth factor deprivation via extracellular signal-regulated kinase-dependent expression of p21 Cip/WAF1

Abstract

Apoptotic regulation of monocytes/macrophages appears to be closely associated with chronic inflammatory reactions. Since it was demonstrated earlier that certain bacterial cell components are involved in apoptotic regulation of these cells, in the present study, we investigated whether the bacterial fimbria, an important cell structure involved in bacterial adherence to host cells, regulates apoptosis of human monocytic THP-1 cells induced under growth factor deprivation. To investigate this point, we used fimbriae of Porphyromonas gingivalis, a pathogen causing periodontal disease, which is a chronic inflammatory disease. The fimbriae inhibited apoptosis of the cells under growth factor deprivation. This inhibitory action of the fimbriae was completely neutralized by anti-fimbrial antibody. The fimbriae stimulated activation of extracellular signal-regulated kinase (ERK) and expression of cyclin-dependent kinase inhibitor p21 Cip/WAF1 (p21) in the cells. The stimulatory effect of the fimbriae on the expression of the p21 protein was inhibited by treatment with PD98059, a specific inhibitor of ERK. The cell apoptosis was inhibited by treatment with Ac-DEVD-CHO, an inhibitor of caspase-3. The fimbriae inhibited the serum withdrawal-induced cleavage of the caspase-3 proform and poly(ADP-ribose) polymerase, one of the caspase-3 substrates. Furthermore, PD98059 and antisense p21 oligonucleotide blocked the fimbrial inhibition of apoptosis and caspase-3 activation of the cells induced by serum withdrawal. These results show that the bacterial fimbriae inhibited apoptosis of THP-1 cells induced under growth factor deprivation via ERK-dependent expression of p21. The present study suggests that bacterial fimbriae act as potent regulators of chronic inflammatory disease, e.g., periodontal disease, through blocking apoptosis of monocytes/macrophages.

FIG. 1

Inhibitory effect of P. gingivalis fimbriae on THP-1 cell apoptosis induced by growth factor deprivation. (A) THP-1 cells were incubated with or without serum in the absence or presence of fimbriae at 5 μg of protein/ml. DNA fragments from the supernatant of the lysed cells were isolated at selected times after the initiation of the cultures and then subjected to agarose gel electrophoresis. φX174 RF DNA HaeIII fragments were used as molecular weight markers (M). (B) The cells were treated as described for panel A, and DNA fragmentation was quantified by the diphenylamine assay. (C) Cells in serum-free medium were incubated in the absence or presence of fimbriae at 5 μg of protein/ml that had been pretreated or not with anti-fimbrial antibody (Ab). DNA fragments from the supernatant of the lysed cells were analyzed by agarose gel electrophoresis at 24 h after the initiation of the cultures. (D) Cells were treated as described for panel C, and DNA fragmentation was quantified by the diphenylamine assay at 24 h after the initiation of the cultures. The results of the diphenylamine assays are expressed as the mean ± standard deviation of three cultures. An identical experiment independently performed gave similar results.

FIG. 2

Inducing effect of P. gingivalis fimbriae on expression of p21 protein in THP-1 cells under growth factor deprivation. THP-1 cells in serum-free medium were pretreated for 24 h in the absence or presence of 1 μM antisense or sense p21 oligonucleotide and were then incubated, also in serum-free medium, in the absence or presence of the fimbriae at 5 μg of protein/ml. The expression of p21 protein in the cells was analyzed by Western blotting with anti-p21 antibody 24 h after the initiation of the fimbrial treatment. An identical experiment independently performed gave similar results.

FIG. 3

Effect of P. gingivalis fimbriae on expression of Bcl-2 protein in THP-1 cells under growth factor deprivation. THP-1 cells were treated as described in the legend to Fig. 2, and the expression of Bcl-2 protein in the cells was analyzed by Western blotting with anti-Bcl-2 antibody 12 and 24 h after the initiation of the cultures. An identical experiment independently performed gave similar results.

FIG. 4

Antisense p21 oligonucleotide blocks fimbrial inhibition of THP-1 cell apoptosis induced under growth factor deprivation. THP-1 cells were treated as described in the legend to Fig. 2, and DNA fragmentation was quantified by the diphenylamine assay 24 h after the initiation of the fimbrial treatment. The results of the assay are expressed as the mean ± standard deviation of three cultures. Variations between values from the fimbria-treated groups with or without antisense p21 oligonucleotide that were found to be statistically significant by Student's t test are indicated by asterisks (∗, P < 0.05; ∗∗, P < 0.01). An identical experiment independently performed gave similar results.

FIG. 5

Effect of P. gingivalis fimbriae on phosphorylation of ERK and MAPK in THP-1 cells under growth factor deprivation. The cells were treated as described in the legend to Fig. 2. The expression of ERK or phosphorylated ERK proteins in the cells was analyzed by Western blotting with anti-p44/42 MAPK antibody or anti-phosphorylated p44/42 MAPK antibody at selected times after the initiation of the cultures. An identical experiment independently performed gave similar results.

FIG. 6

Involvement of ERK and MAPK activity in fimbria-stimulated expression of p21 protein in and inhibitory effect of the fimbriae on THP-1 cell apoptosis induced under growth factor deprivation. (A) THP-1 cells in serum-free medium were pretreated or not for 1 h with PD98059 at 5 μM and then incubated in the absence or presence of fimbriae at 5 μg of protein/ml. After 24 h, the expression of the p21 protein in the cells was analyzed by Western blotting with anti-p21 antibody. (B) Cells were treated as described for panel A, and DNA fragmentation was quantified by the diphenylamine assay 24 h after the initiation of the fimbrial treatment. The results of the assay are expressed as the mean ± standard deviation of three cultures. An identical experiment independently performed gave similar results.

FIG. 7

Effect of caspase inhibitors on THP-1 cell apoptosis induced under growth factor deprivation. THP-1 cells in serum-free medium were treated with the indicated concentration of Ac-YVAD-CHO or Ac-DEVD-CHO or untreated. Then DNA fragmentation was quantified by the diphenylamine assay 24 h after the initiation of the treatment. The results of the assay are expressed as the mean ± standard deviation of three cultures. Similar results were obtained in an identical experiment.

FIG. 8

Inhibitory effect of P. gingivalis fimbria-stimulated ERK and MAPK activity and expression of p21 protein on caspase-3 activation in THP-1 cells induced under growth factor deprivation. (A) THP-1 cells were treated as described in the legend to Fig. 1A. (B) Cells were treated as described in the legend to Fig. 6A. (C) Cells were treated as described in the legend to Fig. 2. After 24 h, the cleavage of the caspase-3 proform and PARP was analyzed by Western blotting with anti-CPP32 antibody or anti-PARP antibody. An identical experiment independently performed gave similar results.