Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010 Graz, Austria.
J Biol Chem. 2012 Jun 15;287(25):21110-20. doi: 10.1074/jbc.M111.319129. Epub 2012 May 3.
Accumulation of palmitic acid (PA) in cells from nonadipose tissues is known to induce lipotoxicity resulting in cellular dysfunction and death. The exact molecular pathways of PA-induced cell death are still mysterious. Here, we show that PA triggers autophagy, which did not counteract but in contrast promoted endothelial cell death. The PA-induced cell death was predominantly necrotic as indicated by annexin V and propidium iodide (PI) staining, absence of caspase activity, low levels of DNA hypoploidy, and an early ATP depletion. In addition PA induced a strong elevation of mRNA levels of ubiquitin carboxyl-terminal hydrolase (CYLD), a known mediator of necroptosis. Moreover, siRNA-mediated knockdown of CYLD significantly antagonized PA-induced necrosis of endothelial cells. In contrast, inhibition and knockdown of receptor interacting protein kinase 1 (RIPK1) had no effect on PA-induced necrosis, indicating the induction of a CYLD-dependent but RIPK1-independent cell death pathway. PA was recognized as a strong and early inducer of autophagy. The inhibition of autophagy by both pharmacological inhibitors and genetic knockdown of the autophagy-specific genes, vacuolar protein sorting 34 (VPS34), and autophagy-related protein 7 (ATG7), could rescue the PA-induced death of endothelial cells. Moreover, the initiation of autophagy and cell death by PA was reduced in endothelial cells loaded with the Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-(acetoxymethyl) ester (BAPTA-AM), indicating that Ca(2+) triggers the fatal signaling of PA. In summary, we introduce an unexpected mechanism of lipotoxicity in endothelial cells and provide several novel strategies to counteract the lipotoxic signaling of PA.
细胞内棕榈酸（PA）的积累已知会诱导脂毒性，导致细胞功能障碍和死亡。PA 诱导细胞死亡的确切分子途径仍然神秘。在这里，我们表明 PA 触发自噬，但没有抵消反而促进了内皮细胞死亡。PA 诱导的细胞死亡主要是坏死，如 Annexin V 和碘化丙啶（PI）染色、无半胱天冬酶活性、低水平的 DNA 亚二倍体和早期 ATP 耗竭所表明的那样。此外，PA 诱导泛素羧基末端水解酶（CYLD）的 mRNA 水平强烈升高，CYLD 是一种已知的坏死介体。此外，siRNA 介导的 CYLD knockdown 显著拮抗了 PA 诱导的内皮细胞坏死。相比之下，抑制和 knockdown 受体相互作用蛋白激酶 1（RIPK1）对 PA 诱导的坏死没有影响，表明诱导了一种依赖 CYLD 但不依赖 RIPK1 的细胞死亡途径。PA 被认为是自噬的强烈和早期诱导剂。通过药理学抑制剂和自噬特异性基因（液泡蛋白分选 34（VPS34）和自噬相关蛋白 7（ATG7）的基因 knockdown 抑制自噬，都可以挽救 PA 诱导的内皮细胞死亡。此外，在用 Ca(2+)螯合剂 1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸-（乙酰氧甲基）酯（BAPTA-AM）加载的内皮细胞中，PA 引发的自噬和细胞死亡减少，表明 Ca(2+)触发了 PA 的致命信号。总之，我们在内皮细胞中引入了一种意想不到的脂毒性机制，并提供了几种新的策略来对抗 PA 的脂毒性信号。
