Apolipoprotein L6, induced in atherosclerotic lesions, promotes apoptosis and blocks beclin 1-dependent autophagy in atherosclerotic cells

Inflammatory cytokine-regulated apoptosis and autophagy play pivotal roles in plaque rupture and thrombosis of atherosclerotic lesions. However, the molecular interplay between apoptosis and autophagy in vascular cells has not been investigated. Our prior study showed that human apolipoprotein L6 (ApoL6), a pro-apoptotic BH3-only member of the Bcl-2 family, was one of the downstream targets of interferon-γ (INFγ), which sensitizes atherosclerotic lesion-derived cells (LDCs) to Fas-induced apoptosis. To investigate whether ApoL6 plays a causal role in atherosclerotic apoptosis and autophagy, in this study, we demonstrate that IFNγ treatment itself strongly induces ApoL6, and ApoL6 is highly expressed and partially colocalized with activated caspase 3 in activated smooth muscle cells in atherosclerotic lesions. In addition, overexpression of ApoL6 promotes reactive oxygen species (ROS) generation, caspase activation, and subsequent apoptosis, which can be blocked by pan caspase inhibitor and ROS scavenger. Knockdown of ApoL6 expression by siApoL6 suppresses INFγ- and Fas-mediated apoptosis. Further, ApoL6 binds Bcl-X L, one of the most abundant anti-death proteins in LDCs. Interestingly, forced ApoL6 expression in LDCs induces degradation of Beclin 1, accumulation of p62, and subsequent attenuation of LC3-II formation and translocation and thus autophagy, whereas siApoL6 treatment reverts the phenotype. Taken together, our results suggest that ApoL6 regulates both apoptosis and autophagy in SMCs. IFNγ-initiated, ApoL6-induced apoptosis in vascular cells may be an important factor causing plaque instability and a potential therapeutic target for treating atherosclerosis and cardiovascular disease. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc

Apolipoprotein L1, a Novel Bcl-2 Homology Domain 3-only Lipid-binding Protein, Induces Autophagic Cell Death*

The Bcl-2 family proteins are important regulators of type I programmed cell death apoptosis; however, their role in autophagic cell death (AuCD) or type II programmed cell death is still largely unknown. Here we report the cloning and characterization of a novel Bcl-2 homology domain 3 (BH3)-only protein, apolipoprotein L1 (apoL1), that, when overexpressed and accumulated intracellularly, induces AuCD in cells as characterized by the increasing formation of autophagic vacuoles and activating the translocation of LC3-II from the cytosol to the autophagic vacuoles. Wortmannin and 3-methyladenine, inhibitors of class III phosphatidylinostol 3-kinase and, subsequently, autophagy, blocked apoL1-induced AuCD. In addition, apoL1 failed to induce AuCD in autophagy-deficient ATG5-/- and ATG7-/- mouse embryonic fibroblast cells, suggesting that apoL1-induced cell death is indeed autophagy-dependent. Furthermore, a BH3 domain deletion construct of apoL1 failed to induce AuCD, demonstrating that apoL1 is a bona fide BH3-only pro-death protein. Moreover, we showed that apoL1 is inducible by p53 in p53-induced cell death and is a lipid-binding protein with high affinity for phosphatidic acid (PA) and cardiolipin (CL). Previously, it has been shown that PA directly interacted with mammalian target of rapamycin and positively regulated the ability of mammalian target of rapamycin to activate downstream effectors. In addition, CL has been shown to activate mitochondria-mediated apoptosis. Sequestering of PA and CL with apoL1 may alter the homeostasis between survival and death leading to AuCD. To our knowledge, this is the first BH3-only protein with lipid binding activity that, when overproduced intracellularly, induces AuCD