The Ca²⁺ concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: significance for the molecular mechanism of Bcl-2 action

Paolo Pinton, Davide Ferrari¹, Elena Rapizzi¹, Francesco Di Virgilio¹, Tullio Pozzan, and Rosario Rizzuto^1,2

Department of Biomedical Sciences and CNR Center for the Study of Biomembranes, University of Padova, Via Colombo 3, I-35121 Padova and ¹Department of Experimental and Diagnostic Medicine, Section of General Pathology and Center for the Study of Inflammatory Diseases, Via Borsari 46, I-44100 Ferrara, Italy ²Corresponding author e-mail: r.rizzuto{at}unife.itP.Pinton and D.Ferrari contributed equally to this work

Abstract: 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 Ca²⁺ concentration in the endoplasmic reticulum ([Ca²⁺]_er) by increasing the passive Ca²⁺ leak from the organelle. To investigate whether this Ca²⁺ depletion is part of the mechanism of action of Bcl-2, we mimicked the Bcl-2 effect on [Ca²⁺]_er by different pharmacological and molecular approaches. All conditions that lowered [Ca²⁺]_er protected HeLa cells from ceramide, a Bcl-2-sensitive apoptotic stimulus, while treatments that increased [Ca²⁺]_er had the opposite effect. Surprisingly, ceramide itself caused the release of Ca²⁺ from the endoplasmic reticulum and thus [Ca²⁺] increased both in the cytosol and in the mitochondrial matrix, paralleled by marked alterations in mitochondria morphology. The reduction of [Ca²⁺]_er levels, as well as the buffering of cytoplasmic [Ca²⁺] changes, prevented mitochondrial damage and protected cells from apoptosis. It is therefore concluded that the Bcl-2-dependent reduction of [Ca²⁺]_er is an important component of the anti-apoptotic program controlled by this oncogene.

Key Words: Keywords: apoptosis/Bcl-2/Ca²⁺/ceramide/mitochondria