BNIP3 and Genetic Control of Necrosis-Like Cell Death through the Mitochondrial Permeability Transition Pore

Abstract

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Δψm) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.

ACKNOWLEDGMENTS

C. Vande Velde and J. Cizeau contributed equally to this study.

We thank Peter Nickerson, Geoff Hicks, Ed Rector, and Guangming Zhong of the University of Manitoba for their assistance with flow cytometry and confocal laser microscopy, as well as Eileen MacMillan-Ward for electron microscopy preparations. We are especially grateful to Emad Alnemri, Guido Kroemer, Yuri Lazebnik, Josef Penninger, Xiaodong Wang, Junying Yuan, and J. A. Duine for their kind gift of reagents and Spencer Gibson for reviewing the manuscript and providing 293-Bcl-2 cells. We also thank Laurie Lange, Elizabeth Henson, and Angela Kemp for their excellent technical assistance and all members of the A.H.G. lab for helpful discussions.

This work was supported by the National Cancer Institute of Canada with funds from the Terry Fox Run and the Medical Research Council of Canada.