Inhibition of phosphatidylinositol 3-kinase sensitizes vascular endothelial cells to cytokine-initiated cathepsin-dependent apoptosis.
In the presence of cycloheximide, tumor necrosis factor or interleukin-1 initiates caspase activation, loss of mitochondrial membrane potential (DeltaPsi), DNA degradation, and nuclear condensation and fragmentation characteristic of apoptotic cell death in human vascular endothelial cells (EC). Inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002, but not inhibition of Akt by dominant-negative mutation, also sensitizes EC to cytokine-initiated apoptosis. Cytokine-initiated caspase activation is slower and comparatively less with LY294002 than with cycloheximide. Cycloheximide but not LY294002 decreases expression of c-FLIP (cellular FLICE inhibitory protein), an inhibitor of caspase-8 activation. The caspase inhibitor zVADfmk completely blocks caspase activation, DNA degradation, and nuclear fragmentation in both cases but only prevents loss of DeltaPsi and cell death for cytokine plus cycloheximide treatment. In contrast, overexpression of Bcl-2 protects EC treated with cytokine plus LY294002 but not EC treated with cytokine plus cycloheximide. The cathepsin B inhibitor CA-074-Me prevents loss of DeltaPsi, caspase activation, and cell death for EC treated with cytokine plus LY294002 but has no effect on EC treated with cytokine plus cycloheximide. Cathepsin B translocates from lysosomes to cytosol following treatment with LY294002 prior to the activation of caspases. These results suggest that inhibition of PI3K allows cytokines to activate a cathepsin-dependent, mitochondrial death pathway in which caspase activation is secondary, is not inhibited by c-FLIP, and is not essential for cell death.
Madge
Lisa A
LA
Interdepartmental Program in Vascular Biology and Transplantation, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Li
Jie-Hui
JH
Choi
Jaehyuk
J
Pober
Jordan S
JS
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000235:genetics; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism; D002467:Cell Nucleus; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology
D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D002401:Cathepsin B; D002867:Chromones; D003513:Cycloheximide; D015853:Cysteine Proteinase Inhibitors; D004791:Enzyme Inhibitors; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D017346:Protein Serine-Threonine Kinases; D011500:Protein Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; C506386:CFLAR protein, human; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C494918:AKT1 protein, human; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human