Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death
Enhancing lysosome biogenesis attenuates BNIP3-induced cardiomyocyte death
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DOI：
10.4161/auto.18658
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发表时间：
2012-03-01
期刊：
影响因子：
13.3
通讯作者：
Diwan, Abhinav
中科院分区：
文献类型：
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作者：
Ma, Xiucui;Godar, Rebecca J.;Diwan, Abhinav
缺氧诱导的促死亡蛋白BNIP 3（BCL-2/腺病毒E1 B 19-kDa相互作用蛋白3）引起线粒体透化，导致缺血再灌注损伤中的心肌细胞死亡。自噬的抑制通过阻止受损线粒体的去除而加速BNIP 3诱导的细胞死亡。我们测试了刺激自噬将减弱BNIP 3诱导的心肌细胞死亡的假设。用BNIP 3（或LacZ作为对照;感染复数= 100）腺病毒转导新生大鼠心肌细胞（NRCM）;并用雷帕霉素（100 nM）刺激自噬。在48小时评估细胞死亡。与对照相比，BNIP 3表达使自噬体丰度增加8倍，并导致心肌细胞死亡增加3.6倍。雷帕霉素处理BNIP 3表达细胞导致自噬体数量进一步增加，而不影响细胞死亡。BNIP 3表达导致自噬体结合的LC 3-II和p62的积累，以及自噬体的增加，但不导致自溶酶体的增加（用双荧光mCherry-GFP-LC 3表达评估）。BNIP 3，而不是跨膜缺失变体，与LC 3相互作用，并与线粒体和溶酶体共定位。然而，BNIP 3并不通过亚细胞分级分离靶向溶酶体，引起溶酶体透化或改变溶酶体pH。相反，BNIP 3诱导的自噬引起溶酶体数量下降，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。转录因子EB（TFEB）在BNIP 3表达细胞中的强制表达增加了溶酶体数量，减少了自噬体并增加了自溶酶体，防止了p62积累，去除了去极化线粒体并减弱了BNIP 3诱导的死亡。我们的结论是，BNIP 3的表达诱导自噬体的积累与心肌细胞中的溶酶体消耗。TFEB（一种溶酶体生物发生因子）的强制表达恢复了自噬体加工并减弱了BNIP 3诱导的细胞死亡。
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.