A constitutive cytoprotective pathway protects endothelial cells from lipopolysaccharide-induced apoptosis.
Lipopolysaccharide (LPS) has been implicated as the bacterial component responsible for much of the endothelial cell injury/dysfunction associated with Gram-negative bacterial infections. Protein synthesis inhibition is required to sensitize the endothelium to lipopolysaccharide-induced apoptosis, suggesting that a constitutive or inducible cytoprotective protein(s) is required for endothelial survival. We have identified two known endothelial anti-apoptotic proteins, c-FLIP and Mcl-1, the expression of which is decreased markedly in the presence of cycloheximide. Decreased expression of both proteins preceded apoptosis evoked by lipopolysaccharide + cycloheximide. Caspase inhibition protected against apoptosis, but not the decreased expression of c-FLIP and Mcl-1, suggesting that they exert protection upstream of caspase activation. Inhibition of the degradation of these two proteins with the proteasome inhibitor, lactacystin, prevented lipopolysaccharide + cycloheximide-induced apoptosis. Similarly, lactacystin protected against endothelial apoptosis induced by either tumor necrosis factor-alpha or interleukin-1beta in the presence of cycloheximide. That apoptosis could be blocked in the absence of new protein synthesis by inhibition of the proteasome degradative pathway implicates the requisite involvement of a constitutively expressed protein(s) in the endothelial cytoprotective pathway. Finally, reduction of FLIP expression with antisense oligonucleotides sensitized endothelial cells to LPS killing, demonstrating a definitive role for FLIP in the protection of endothelial cells from LPS-induced apoptosis.
Bannerman
D D
DD
Department of Medicine, University of Washington School of Medicine, Seattle, Washington 98104, USA. dbannerm@u.washington.edu
Tupper
J C
JC
Ricketts
W A
WA
Bennett
C F
CF
Winn
R K
RK
Harlan
J M
JM
D017209:Apoptosis / Q000187:drug effects*; D001483:Base Sequence; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000187:drug effects; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D006868:Hydrolysis; D007375:Interleukin-1 / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009097:Multienzyme Complexes / Q000187:drug effects; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B / Q000502:physiology; D009363:Neoplasm Proteins / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; D017931:DNA Primers; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D008070:Lipopolysaccharides; D009097:Multienzyme Complexes; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D016328:NF-kappa B; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex