h-89 and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

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.

Topics: Adhesins, Bacterial; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Inducing Factor; Caspase 2; Caspase 3; Caspases; Cysteine Endopeptidases; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fluorescent Antibody Technique; Gingipain Cysteine Endopeptidases; Humans; Immunoblotting; In Situ Nick-End Labeling; Isoquinolines; Porphyromonas gingivalis; RNA, Small Interfering; Signal Transduction; Sulfonamides; Tumor Necrosis Factor-alpha

Glucagon and the glucagon-like peptides regulate metabolic functions via signaling through a glucagon receptor subfamily of G protein-coupled receptors. Activation of glucagon-like peptide-2 receptor (GLP-2R) signaling maintains the integrity of the intestinal epithelial mucosa via regulation of crypt cell proliferation. Because GLP-2 decreases mortality and reduces intestinal apoptosis in rodents after experimental injury, we examined whether GLP-2R signaling directly modifies the cellular response to external injury. We show here that activation of GLP-2R signaling inhibits cycloheximide-induced apoptosis in baby hamster kidney fibroblasts expressing a transfected GLP-2 receptor. GLP-2 reduced DNA fragmentation and improved cell survival, in association with reduced activation of caspase-3 and decreased poly(ADP-ribose) polymerase cleavage and reduced caspase-8 and caspase-9-like activities. Both GLP-2 and forskolin reduced mitochondrial cytochrome c release and decreased the cycloheximide-induced cleavage of caspase-3 in the presence or absence of the PKA inhibitor H-89. Similarly, GLP-2 increased cell survival following cycloheximide in the presence of the kinase inhibitors PD98054 and LY294002. These findings provide evidence that signaling through G protein-coupled receptors of the glucagon superfamily is directly linked to regulation of apoptosis and suggest the existence of a cAMP-dependent protein kinase-, phosphatidylinositol 3-kinase-, and mitogen-activated protein kinase-independent pathway coupling GLP-2R signaling to caspase inhibition and cell survival.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Division; Cell Line; Cell Survival; Chromones; Colforsin; Cricetinae; Cyclic AMP-Dependent Protein Kinases; Cycloheximide; Cysteine Proteinase Inhibitors; Cytochrome c Group; Cytosol; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Activation; Enzyme Inhibitors; Epithelium; Fibroblasts; Flavonoids; Glucagon-Like Peptide 2; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Intestinal Mucosa; Isoquinolines; Microscopy, Fluorescence; Mitochondria; Morpholines; Peptides; Poly(ADP-ribose) Polymerases; Protein Synthesis Inhibitors; Receptors, Cell Surface; Receptors, Glucagon; Signal Transduction; Sulfonamides; Time Factors; Transfection