Trypsin-like protease-active extracellular protein extracts from Porphyromonas gingivalis ATCC 33277 induce apoptosis in bovine aortic endothelial cells.
OBJECTIVE
Certain virulence factors participating in periodontitis may relate to cardiovascular diseases. This study was to evaluate the pro-apoptotic effect of protein extracts from Porphyromonas gingivalis on bovine aortic endothelial cells (BAECs).


METHODS
The BAECs were exposed to trypsin-like protease-active protein extracts from P. gingivalis, and apoptosis was examined by Hoechst 33342 staining, DNA fragmentation assay and cleaved caspase-3 detection. When BAECs were exposed to protein extracts pretreated with trypsin-like protease inhibitor (TLCK), the apoptosis rate was evaluated by Annexin V-propidium iodide staining. To further study the potential mechanism of the pro-apoptotic effect, immunoblotting was used to detect expression of alpha-tubulin, integrin beta1 and activated ERK1/2 in BAECs treated with protein extracts or cultured in suspension.


RESULTS
After exposure to the protein extracts, BAECs exhibited loss of cell adhesion and apoptotic cell death. The pro-apoptotic effect could be delayed by TLCK pretreatment. In addition, BAECs treated with protein extracts showed decreased levels of alpha-tubulin, integrin beta1 and activated ERK1/2. When BAECs were cultured in suspension, ERK1/2 activation was also inhibited, but the percentage decrease in ERK1/2 activation was less than that induced by protein extracts. Moreover, no significantly altered expression of alpha-tubulin was detected in suspended cells.


CONCLUSIONS
Trypsin-like protease-active protein extracts from P. gingivalis could induce apoptosis of BAECs. The destruction of alpha-tubulin and integrin beta1 and decrease of ERK1/2 activation might contribute to the pro-apoptotic effect of the protein extracts.
Tian
N
N
Department of Periodontology, Peking University School and Hospital of Stomatology, Beijing, China.
Ouyang
X Y
XY
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects*; D017209:Apoptosis / Q000187:drug effects; D001426:Bacterial Proteins / Q000494:pharmacology*; D002417:Cattle; D002448:Cell Adhesion / Q000187:drug effects; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D042783:Endothelial Cells / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D019012:Integrin beta1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D014357:Trypsin / Q000494:pharmacology*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D014404:Tubulin / Q000378:metabolism; D037521:Virulence Factors / Q000494:pharmacology
D001426:Bacterial Proteins; D017077:Culture Media, Conditioned; D019012:Integrin beta1; D014361:Trypsin Inhibitors; D014404:Tubulin; D037521:Virulence Factors; D020928:Mitogen-Activated Protein Kinases; D014357:Trypsin