Ionomycin-activated Calpain Triggers Apoptosis

Abstract

Ubiquitous calpains (-and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x L at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization

“…[14][15][16][17]33,34 Upon activation, CAPNs cleave apoptosisrelated proteins, triggering cytochrome c release from mitochondria and inducing caspase activation. 12 Our data demonstrate that CAPN1 is already localized in the Representative western blot analysis of at least three independent biological samples showing calpain-1 and -2, Lamp2a and total Lamp2 (tLamp) in control lactating (0 h), 72-h-weaned mice and 72-h-weaned mice in which calpain-1 was knocked down by specific siRNA treatment (siRNA calpain-1). a-Tubulin was used as loading control Role of calpains in mammary gland involution T Arnandis et al mitochondria during lactation (Figure 3a) and becomes activated during involution, releasing cytochrome c (Figure 3d).…”

“…CAPNs have been implicated in apoptosis, 12,13 being involved in the mitochondrial pathway. In this sense, Bcl-2 and Bid are cleaved by activated CAPNs, promoting cytochrome c release and induction of the intrinsic apoptotic pathway.…”

“…In this sense, Bcl-2 and Bid are cleaved by activated CAPNs, promoting cytochrome c release and induction of the intrinsic apoptotic pathway. 12 Besides, although CAPNs are considered cytosolic proteases, CAPN1 is enriched in the mitochondrial fraction.…”

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

“…There are two ubiquitous forms, calpain I (-calpain) and calpain II (m-calpain) that are activated by micromolar and millimolar concentrations of Ca 2ϩ in vitro, respectively, as well as a number of tissue-specific isoforms. Several members of the Bcl-2 family of cell death regulators are processed by calpain (3), and calpain cleavage of Bid has been implicated in mitochondrial permeabilization and cell death following ischemia/reperfusion in the heart (4,5). Although the sequence specificity of calpain proteolysis is incompletely understood and may depend in large part on the tertiary structure of the target protein, some calpain substrates are distinguished by a PEST sequence rich in proline (P), glutamate (E) (or aspartate), and serine (S)/threonine (T) residues (6,7).…”

Involvement of Intracellular Ca2+ Levels in Nonsteroidal Anti-inflammatory Drug-induced Apoptosis

“…For example, many apoptotic stimuli, such as activation of surface antigen receptors, increase the intracellular Ca 2ϩ level, and compounds that directly increase the intracellular Ca 2ϩ level (Ca 2ϩ ionophores and inhibitors for sarcoendoplasmic reticulum Ca 2ϩ ATPase (SERCA)) have been shown to induce apoptosis (23)(24)(25). Various mechanisms have been proposed for the Ca 2ϩ -induced apoptosis pathway, such as activation of protein kinase C, opening of permeability transition pores in mitochondria, and stimulation of reactive oxygen species synthesis (26 -29).…”