Cross-talk between calpain and caspase proteolytic systems during neuronal apoptosis.
Cross-talk between calpain and caspase proteolytic systems has complicated efforts to determine their distinct roles in apoptotic cell death. This study examined the effect of overexpressing calpastatin, the specific endogenous calpain inhibitor, on the activity of the two proteolytic systems following an apoptotic stimulus. Human SH-SY5Y neuroblastoma cells were stably transfected with full-length human calpastatin cDNA resulting in 20-fold overexpression based on Western blot and 5-fold greater calpain inhibitory activity in cell extracts. Wild type and calpastatin overexpressing (CST1) cells were neuronally differentiated and apoptosis-induced with staurosporine (0.1-1.0 microm). Calpastatin overexpression decreased calpain activation, increased caspase-3-like activity, and accelerated the appearance of apoptotic nuclear morphology. Following 0.1-0.2 microm staurosporine, plasma membrane integrity based on calcein-acetoxymethyl fluorescence was significantly greater at 24 h in differentiated CST1 compared with differentiated wild type cells. However, this protective effect was lost at higher staurosporine doses (0.5-1.0 microm), which resulted in pronounced caspase-mediated degradation of the overexpressed calpastatin. These results suggest a dual role for calpains during neuronal apoptosis. In the early execution phase, calpain down-regulates caspase-3-like activity and slows progression of apoptotic nuclear morphology. Subsequent calpain activity, facilitated by caspase-mediated degradation of calpastatin, contributes to plasma membrane disruption and secondary necrosis.
Neumar
Robert W
RW
Department of Emergency Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104, USA. rneumar@mail.med.upenn.edu
Xu
Y Anne
YA
Gada
Hemal
H
Guttmann
Rodney P
RP
Siman
Robert
R
D017209:Apoptosis; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008856:Microscopy, Fluorescence; D009336:Necrosis; D009474:Neurons / Q000473:pathology*; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; C033668:calpastatin; C505407:CASP3 protein, human; D002154:Calpain; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine