Calpain-1 regulates Bax and subsequent Smac-dependent caspase-3 activation in neutrophil apoptosis.
In the absence and in the resolution of inflammatory responses, neutrophils rapidly undergo spontaneous apoptosis. Here we report about a new apoptosis pathway in these cells that requires calpain-1 activation and is essential for the enzymatic activation of the critical effector caspase-3. Decreased levels of calpastatin, a highly specific intrinsic inhibitor of calpain, resulted in activation of calpain-1, but not calpain-2, in neutrophils undergoing apoptosis, a process that was blocked by a specific calpain-1 inhibitor or by intracellular delivery of a calpastatin peptide. Further support for the importance of the calpastatin-calpain system was obtained by analyzing neutrophils from patients with cystic fibrosis that exhibited delayed apoptosis, associated with markedly increased calpastatin and decreased calpain-1 protein levels compared with neutrophils from control individuals. Additional studies were designed to place calpain-1 into the hierarchy of biochemical events leading to neutrophil apoptosis. Pharmacological calpain inhibition during spontaneous and Fas receptor-induced neutrophil apoptosis prevented cleavage of Bax into an 18-kDa fragment unable to interact with Bcl-xL. Moreover, calpain blocking prevented the mitochondrial release of cytochrome c and Smac, which was indispensable for caspase-3 processing and enzymatic activation, both in the presence and absence of agonistic anti-Fas receptor antibodies. Taken together, calpastatin and calpain-1 represent critical proximal elements in a cascade of pro-apoptotic events leading to Bax, mitochondria, and caspase-3 activation, and their altered expression appears to influence the life span of neutrophils under pathologic conditions.
Altznauer
Frank
F
Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland.
Conus
Sébastien
S
Cavalli
Andrea
A
Folkers
Gerd
G
Simon
Hans-Uwe
HU
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D002154:Calpain / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000737:chemistry / Q000378:metabolism*; D016923:Cell Death; D002478:Cells, Cultured; D003550:Cystic Fibrosis / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D003720:Densitometry; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D018613:Microscopy, Confocal; D024101:Mitochondrial Proteins / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009504:Neutrophils / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions; D013997:Time Factors; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019014:fas Receptor / Q000737:chemistry
D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; C412403:DIABLO protein, human; D047908:Intracellular Signaling Peptides and Proteins; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D019014:fas Receptor; C033668:calpastatin; D045304:Cytochromes c; C505407:CASP3 protein, human; D002154:Calpain; D053148:Caspase 3; D020169:Caspases