Abstract
Porphyromonas gingivalis is an oral bacterium that causes pathology in a number of dental infections that are associated with increased fibroblast cell death. Studies presented here demonstrated that P. gingivalis stimulates cell death by apoptosis rather than necrosis. Unlike previous studies apoptosis was induced independent of proteolytic activity and was also independent of caspase activity because a pancaspase inhibitor, Z-VAD-fmk, had little effect. Moreover, P. gingivalis downregulated caspase-3 mRNA levels and caspase-3 activity. The consequence of this downregulation was a significant reduction in tumour necrosis factor-alpha-induced apoptosis, which is caspase-3-dependent. Immunofluorescence and immunoblot analysis revealed P. gingivalis-induced translocation of apoptosis-inducing factor (AIF) from the cytoplasm to the nucleus. siRNA studies were undertaken and demonstrated that P. gingivalis stimulated cell death was significantly reduced when AIF was silenced (P < 0.05). Treatment of human gingival fibroblasts with H-89, a protein kinase A inhibitor that blocks AIF activation also reduced P. gingivalis-induced apoptosis (P < 0.05). These results indicate that P. gingivalis causes fibroblast apoptosis through a pathway that involves protein kinase A and AIF, is not dependent upon bacterial proteolytic activity and is also independent of the classic apoptotic pathways involving caspase-3.
Publication types
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Research Support, N.I.H., Extramural
MeSH terms
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Adhesins, Bacterial / genetics
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Adhesins, Bacterial / metabolism
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Amino Acid Chloromethyl Ketones / pharmacology
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Apoptosis / drug effects
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Apoptosis / physiology*
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Apoptosis Inducing Factor / genetics
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Apoptosis Inducing Factor / metabolism*
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Apoptosis Inducing Factor / physiology
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Caspase 2 / genetics
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Caspase 2 / metabolism
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Caspase 3 / genetics
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Caspase 3 / metabolism
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Caspases / genetics
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Caspases / metabolism
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Cysteine Endopeptidases / genetics
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Cysteine Endopeptidases / metabolism
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Enzyme-Linked Immunosorbent Assay
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Fibroblasts / cytology
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Fibroblasts / metabolism*
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Fibroblasts / microbiology
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Fluorescent Antibody Technique
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Gingipain Cysteine Endopeptidases
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Humans
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Immunoblotting
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In Situ Nick-End Labeling
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Isoquinolines / pharmacology
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Porphyromonas gingivalis / genetics
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Porphyromonas gingivalis / growth & development*
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Porphyromonas gingivalis / metabolism
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RNA, Small Interfering / genetics
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Sulfonamides / pharmacology
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Tumor Necrosis Factor-alpha / pharmacology
Substances
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Adhesins, Bacterial
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Amino Acid Chloromethyl Ketones
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Apoptosis Inducing Factor
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Gingipain Cysteine Endopeptidases
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Isoquinolines
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RNA, Small Interfering
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Sulfonamides
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Tumor Necrosis Factor-alpha
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benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone
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Caspase 2
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Caspase 3
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Caspases
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Cysteine Endopeptidases
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N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide