Center for Cardiovascular Research, Division of Cardiology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.
Autophagy. 2012 Mar;8(3):297-309. doi: 10.4161/auto.18658. Epub 2012 Feb 3.
Hypoxia-inducible pro-death protein BNIP3 (BCL-2/adenovirus E1B 19-kDa interacting protein 3), provokes mitochondrial permeabilization causing cardiomyocyte death in ischemia-reperfusion injury. Inhibition of autophagy accelerates BNIP3-induced cell death, by preventing removal of damaged mitochondria. We tested the hypothesis that stimulating autophagy will attenuate BNIP3-induced cardiomyocyte death. Neonatal rat cardiac myocytes (NRCMs) were adenovirally transduced with BNIP3 (or LacZ as control; at multiplicity of infection = 100); and autophagy was stimulated with rapamycin (100 nM). Cell death was assessed at 48 h. BNIP3 expression increased autophagosome abundance 8-fold and caused a 3.6-fold increase in cardiomyocyte death as compared with control. Rapamycin treatment of BNIP3-expressing cells led to further increase in autophagosome number without affecting cell death. BNIP3 expression led to accumulation of autophagosome-bound LC3-II and p62, and an increase in autophagosomes, but not autolysosomes (assessed with dual fluorescent mCherry-GFP-LC3 expression). BNIP3, but not the transmembrane deletion variant, interacted with LC3 and colocalized with mitochondria and lysosomes. However, BNIP3 did not target to lysosomes by subcellular fractionation, provoke lysosome permeabilization or alter lysosome pH. Rather, BNIP3-induced autophagy caused a decline in lysosome numbers with decreased expression of the lysosomal protein LAMP-1, indicating lysosome consumption and consequent autophagosome accumulation. Forced expression of transcription factor EB (TFEB) in BNIP3-expressing cells increased lysosome numbers, decreased autophagosomes and increased autolysosomes, prevented p62 accumulation, removed depolarized mitochondria and attenuated BNIP3-induced death. We conclude that BNIP3 expression induced autophagosome accumulation with lysosome consumption in cardiomyocytes. Forced expression of TFEB, a lysosomal biogenesis factor, restored autophagosome processing and attenuated BNIP3-induced cell death.
缺氧诱导的促死亡蛋白 BNIP3（BCL-2/腺病毒 E1B 19kDa 相互作用蛋白 3），在缺血再灌注损伤中引发线粒体通透性增加，导致心肌细胞死亡。自噬的抑制通过阻止受损线粒体的清除而加速 BNIP3 诱导的细胞死亡。我们检验了这样一个假说，即刺激自噬将减轻 BNIP3 诱导的心肌细胞死亡。用腺病毒转导 BNIP3（或 LacZ 作为对照；感染复数=100）转染新生大鼠心肌细胞（NRCM），并用雷帕霉素（100 nM）刺激自噬。在 48 小时评估细胞死亡。BNIP3 的表达增加了自噬体的丰度 8 倍，并使心肌细胞死亡增加了 3.6 倍，与对照相比。雷帕霉素处理 BNIP3 表达细胞导致自噬体数量进一步增加，而不影响细胞死亡。BNIP3 的表达导致自噬体结合 LC3-II 和 p62 的积累增加，以及自噬体的增加，但不包括自溶体（通过双重荧光 mCherry-GFP-LC3 表达来评估）。BNIP3 与 LC3 相互作用并与线粒体和溶酶体共定位，但不是跨膜缺失变体，而不是通过亚细胞分级分离靶向溶酶体，引发溶酶体通透性或改变溶酶体 pH。相反，BNIP3 诱导的自噬导致溶酶体数量减少，溶酶体蛋白 LAMP-1 表达减少，表明溶酶体消耗和随后的自噬体积累。在 BNIP3 表达细胞中强制表达转录因子 EB（TFEB）增加了溶酶体数量，减少了自噬体并增加了自溶体，防止了 p62 的积累，去除了去极化的线粒体，并减轻了 BNIP3 诱导的死亡。我们的结论是，BNIP3 的表达诱导了心肌细胞中自噬体的积累和溶酶体的消耗。强制表达溶酶体生物发生因子 TFEB 恢复了自噬体的处理并减轻了 BNIP3 诱导的细胞死亡。
