Requirement of Caspase-3 for Apoptotic Calcium Release

Abstract

Calcium is known to be an important second messenger in regulation of various physiological processes including execution of programmed cell death. Calcium release from endoplasmic reticulum (ER) triggers activation of multiple pro-apoptotic factors resulting in apoptosis progression and, ultimately, cell death. Apoptotic calcium release from ER stores is mediated by inositol 1,4,5-trisphosphate receptor (IP3R) channels. Different models have confirmed the importance of IP3R channels in modulation of apoptosis, however many details of IP3R-dependent calcium release still remain enigmatic. Previously, we have demonstrated that calcium release from IP3R channels in response to apoptotic stimuli occurs in two temporally distinct phases. A rapid oscillatory cytosolic calcium release during initiation of apoptotic signaling is followed by delayed and sustained increase in calcium levels associated with persistent opening of IP3R channels. Here we show that early elevations in cytosolic calcium do not require caspase-3 activity. Using MCF-7 cells deficient in caspase-3, we detected robust increase in calcium levels in response to staurosporine treatment indicating that calcium release during initiation of apoptosis occurs independently of caspase-3. To further determine contribution of caspase-3 in regulation of calcium release we generated a MCF-7 cell line stably expressing pro-caspase-3. Additionally, we used TAT-based transducing peptide to deliver active recombinant caspase-3 directly into living MCF-7 cells. Utilizing both methodologies, we found that caspase-3 has a marginal effect on the early events leading to cytosolic calcium elevations and irreversible commitment to apoptotic cell death. These results suggest that caspase-3 mediated truncation of IP3R channels is a consequence, not causative, of apoptotic signaling events.