DT40 cells lacking the Ca ²⁺ -binding protein annexin 5 are resistant to Ca ²⁺ -dependent apoptosis

Abstract

Annexins are widely expressed Ca 2؉ -dependent phospholipidbinding proteins with poorly understood physiological roles. Proposed functions include Ca 2؉ channel activity and vesicle trafficking, but neither have been proven in vivo. Here we used targeted gene disruption to generate B-lymphocytes lacking annexin 5 (Anx5) expression and show that this results in reduced susceptibility to a range of apoptotic stimuli. By comparison B-lymphocytes lacking annexin 2 (Anx2) showed no such resistance, providing evidence that this effect is specific to loss of Anx5. The defect in the ANX5 ؊/؊ cells occurs early in the apoptotic program before nuclear condensation, caspase 3 activation, and cell shrinkage, but downstream of an initial Ca 2؉ influx. Only UVA͞B irradiation induced similar levels of apoptosis in wild-type and ANX5 ؊/؊ cells. Unexpectedly, ANX5 ؊/؊ cells permeabilized in vitro also failed to release mitochondrial cytochrome C, suggesting a possible mechanism for their resistance to apoptosis. These findings demonstrate a role for Anx5 in determining the susceptibility of B-lymphocytes to apoptosis.

Gonadotropin Releasing Hormone (GnRH) Enhances Annexin A5 mRNA Expression through Mitogen Activated Protein Kinase (MAPK) in L.BETA.T2 Pituitary Gonadotrope Cells

“…These activities are thought to be due to its ability to bind phospholipids, although it is not clear whether these activities are relevant to physiological function. However, as we showed in the gonadotropes, annexin A5 may be at least necessary for an intracellular signaling in some cell types [14][15][16]. …”

“…showed that annexin A5 knock-out lymphocytes become resistant to various apoptotic stimuli [16]. Although these findings do not form a single clear picture, most reports indicate that annexin A5 participates in protein-protein signaling complexes.…”

Ways of dying: multiple pathways to apoptosis

“…Although Ca 2+ ions seem to signal between the ER and mitochondria, whether or how this promotes caspase activation is obscure (see below). One potential mediator of Ca 2+ signaling is the Ca 2+ -binding annexin-5, recently shown to be required for apoptosis in response to certain insults (Hawkins et al 2002). Because cells lacking annexin-5 failed to release cytochrome c from mitochondria, and chelators of Ca 2+ ions protected wild-type cells, annexin-5 might transduce a critical Ca 2+ signal from the ER needed for mitochondrial disruption.…”

Annexins – unique membrane binding proteins with diverse functions

“…Other annexin-knockout models (mice lacking annexins A1, A2, A5 or A6, and DT40 chicken pre-B cells lacking annexin A5) also show no obvious phenotype related to a primary defect in vesicle docking and/or fusion events (Brachvogel et al, 2003;Hannon et al, 2003;Hawkins et al, 1999;Hawkins et al, 2002;Ling et al, 2004;Song et al, 2002). This indicates that the annexins targeted in these mice do not serve as essential factors in vesicle docking and/or fusion or that such functions are redundant or taken over by another member of the family during mouse development.…”