Matrix regulation of skeletal cell apoptosis III: mechanism of ion pair-induced apoptosis.
Our previous work has demonstrated that while the Ca(2+) and Pi ions acting in concert function as a potent osteoblast apoptogen, the underlying mechanisms by which it activates cell death is not known. We hypothesize that the ion pair causes release of Ca(2+) from intracellular stores ([Ca(2+)]i); the increase in intracellular calcium prompts the mitochondria to uptake more calcium. This accumulation of calcium eventually results in the loss of mitochondrial membrane potential (MMP) and, subsequently, apoptosis. To test this hypothesis, we evaluated apoptosome formation in MC3T3-E1 osteoblast-like cells treated with the ion pair. Western blot analysis indicated migration of cytochrome-c and Smac/DIABLO from mitochondria to the cytoplasm. Inhibition of either the electron transfer chain (with antimycin a and rotenone), or the activation of a MMP transition (with bongkrekic acid) inhibited apoptosis in a dose-dependent manner. Pre-treating osteoblasts with ruthenium red, a Ca(2+) uniporter inhibitor of both mitochondria and the endoplasmic reticulum (ER), also completely abolished Ca(2+.)Pi-induced apoptosis. Moreover, we showed that an increase in [Ca(2+)]i preceded the increase in MMP over the first 45 min of treatment; a mitochondrial membrane permeability transition was evident at 75 min. To determine the role of ER, Ca(2+) stores in the generation of the apoptotic signal by the ion pair, cells were treated with several inhibitors. Apoptosis was inhibited when cells were treated with dantrolene, an inhibitor of ER ryanodine receptors, and 2-aminodiphenylborate, an IP3 Ca(2+) channel inhibitor, but not cyclopiazonic acid, an ER Ca(2)-ATPase inhibitor. Together, these data demonstrate that Ca(2+) Pi-induced osteoblast apoptosis is characterized by the generation of an apoptosome and that Ca(2+) release from ER stores may promote ion pair-dependent cell death.
Saunders
Ray
R
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107-5099, USA.
Szymczyk
Krysia H
KH
Shapiro
Irving M
IM
Adams
Christopher S
CS
D016475:3T3 Cells; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005109:Extracellular Matrix / Q000502:physiology*; D006868:Hydrolysis; D008564:Membrane Potentials; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000502:physiology; D024101:Mitochondrial Proteins / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000201:enzymology; D010710:Phosphates / Q000378:metabolism*
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C499605:Diablo protein, mouse; D024101:Mitochondrial Proteins; D010710:Phosphates; D045304:Cytochromes c; D020169:Caspases; D002118:Calcium