The Ca(2+) concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action

The mechanism of action of the anti-apoptotic oncogene Bcl-2 is still largely obscure. We have recently shown that the overexpression of Bcl-2 in HeLa cells reduces the Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](er)) by increasing the passive Ca(2+) leak from the organelle. To investigate whether this Ca(2+) depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca(2+)](er) by different pharmacological and molecular approaches. All conditions that lowered [Ca(2+)](er) protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca(2+)](er) had the opposite effect. Surprisingly, ceramide itself caused the release of Ca(2+) from the endoplasmic reticulum and thus [Ca(2+)] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca(2+)](er) levels, as well as the buffering of cytoplasmic [Ca(2+)] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca(2+)](er) is an important component of the anti-apoptotic program controlled by this oncogene.