Evidence that BCL-2 represses apoptosis by regulating endoplasmic reticulum-associated Ca²⁺ fluxes

BCL-2 is a 26-kDa integral membrane protein that represses apoptosis by an unknown mechanism. Recent findings indicate that Ca²⁺ release from the endoplasmic reticulum (ER) mediates apoptosis in mouse lymphoma cells. In view of growing evidence that BCL-2 localizes to the ER, as well as mitochondria and the perinuclear membrane, we investigated the possibility that BCL-2 represses apoptosis by regulating Ca²⁺ fluxes through the ER membrane. A cDNA encoding BCL-2 was introduced into WEHI7.2 cells and two subclones, W.Hb12 and W.Hb13, which express high and low levels of BCL-2 mRNA and protein, respectively, were isolated. WEHI7.2 cells underwent apoptosis in response to treatment with the glucocorticoid hormone dexamethasone, whereas W.Hb12 and W.Hb13 cells were protected from apoptosis, revealing a direct relationship between the level of BCL-2 expression and the degree of protection. Significantly, BCL-2 also blocked induction of apoptosis by thapsigargin (TG), a highly specific inhibitor of the ER-associated Ca²⁺ pump. TG completely inhibited ER Ca²⁺ pumping in both WEHI7.2 and W.Hb12 cells, but the release of Ca²⁺ into the cytosol after inhibition of ER Ca²⁺ pumping was significantly less in W.Hb12 cells than in WEHI7.2 cells, indicating that BCL-2 reduces Ca²⁺ efflux through the ER membrane. By reducing ER Ca²⁺ efflux, BCL-2 interfered with a signal for "capacitative" entry of extracellular Ca²⁺, preventing a sustained increase of cytosolic Ca²⁺ in TG-treated cells. These findings suggest that BCL-2 either directly or indirectly regulates the flux of Ca²⁺ across the ER membrane, thereby abrogating Ca²⁺ signaling of apoptosis.