ABSTRACT

Lipotoxicity, induced by saturated fatty acid (SFA)-mediated cell death, plays an important role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). The KEAP1 (kelch like ECH associated protein 1)-NFE2L2/NRF2 (nuclear factor, erythroid 2 like 2) pathway is a pivotal defense mechanism against lipotoxicity. We previously reported that SQSTM1/p62 has a cytoprotective role against lipotoxicity through activation of the noncanonical KEAP1- NFE2L2 pathway in hepatocytes. However, the underlying mechanisms and physiological relevance of this pathway have not been clearly defined. Here, we demonstrate that NFE2L2-mediated induction of SQSTM1 activates the noncanonical KEAP1-NFE2L2 pathway under lipotoxic conditions. Furthermore, we identified that SQSTM1 induces ULK1 (unc-51 like autophagy activating kinase 1) phosphorylation by facilitating the interaction between AMPK (AMP-activated protein kinase) and ULK1, leading to macroautophagy/autophagy induction, followed by KEAP1 degradation and NFE2L2 activation. Accordingly, the activity of this SQSTM1-mediated noncanonical KEAP1-NFE2L2 pathway conferred hepatoprotection against lipotoxicity in the livers of conventional sqstm1- and liver-specific sqstm1-knockout mice. Moreover, this pathway activity was evident in the livers of patients with nonalcoholic fatty liver. This axis could thus represent a novel target for NAFLD treatment.

Abbreviations: ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; BafA1: bafilomycin A₁; CM-H2DCFDA:5-(and-6)-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate; CQ: chloroquine; CUL3: cullin 3; DMSO: dimethyl sulfoxide; FASN: fatty acid synthase; GSTA1: glutathione S-transferase A1; HA: hemagglutinin; Hepa1c1c7: mouse hepatoma cells; HMOX1/HO-1: heme oxygenase 1; KEAP1: kelch like ECH associated protein 1; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTORC1: mechanistic target of rapamycin kinase complex 1; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NAC: N-acetyl-L-cysteine; NAFLD: nonalcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; NFE2L2/NRF2: nuclear factor, erythroid 2 like 2; NQO1: NAD(P)H quinone dehydrogenase 1; PA: palmitic acid; PARP: poly (ADP-ribose) polymerase 1; PRKAA1/2: protein kinase AMP-activated catalytic subunits alpha1/2; RBX1: ring-box 1; ROS: reactive oxygen species; SESN2: sestrin 2; SFA: saturated fatty acid; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; TBK1: TANK binding kinase 1; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; ULK1: unc-51 like autophagy activating kinase.

摘要

由饱和脂肪酸 (SFA) 介导的细胞死亡诱导的脂毒性在非酒精性脂肪性肝病 (NAFLD) 的发病机制中起重要作用。KEAP1（Kelch 样 ECH 相关蛋白 1）-NFE2L2/NRF2（核因子，类红细胞 2 样 2）通路是对抗脂毒性的关键防御机制。我们之前报道过，SQSTM1/p62 通过激活肝细胞中的非经典 KEAP1-NFE2L2 通路，具有抗脂毒性的细胞保护作用。然而，该途径的潜在机制和生理相关性尚未明确定义。在这里，我们证明了 NFE2L2 介导的 SQSTM1 诱导在脂毒性条件下激活了非规范的 KEAP1-NFE2L2 通路。此外，我们发现 SQSTM1 通过促进 AMPK（AMP 激活蛋白激酶）和 ULK1 之间的相互作用来诱导 ULK1（unc-51 类似自噬激活激酶 1）磷酸化，导致巨自噬/自噬诱导，然后是 KEAP1 降解和 NFE2L2 激活。因此，这种 SQSTM1 介导的非经典 KEAP1-NFE2L2 通路的活性赋予了对常规肝脏中脂毒性的保肝作用。sqstm1 - 和肝脏特异性sqstm1基因敲除小鼠。此外，该途径活性在非酒精性脂肪肝患者的肝脏中很明显。因此，该轴可以代表 NAFLD 治疗的新目标。

缩写： ACACA：乙酰辅酶A羧化酶α；ACTB：肌动蛋白β；BafA1：巴弗洛霉素 A ₁; CM-H2DCFDA:5-(and-6)-chloromethyl-2ʹ,7ʹ-dichlorodihydrofluorescein diacetate；CQ：氯喹；CUL3：cullin 3；DMSO：二甲亚砜；FASN：脂肪酸合成酶；GSTA1：谷胱甘肽S-转移酶A1；HA：血凝素；Hepa1c1c7：小鼠肝癌细胞；HMOX1/HO-1：血红素加氧酶 1；KEAP1: kelch 样 ECH 相关蛋白 1；MAP1LC3B/LC3B：微管相关蛋白1轻链3；MEF：小鼠胚胎成纤维细胞；MTORC1：雷帕霉素激酶复合物 1 的机制靶点；MTT：3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑；NAC：N-乙酰基-L-半胱氨酸；NAFLD：非酒精性脂肪肝；NASH：非酒精性脂肪性肝炎；NFE2L2/NRF2：核因子，类红细胞2像2；NQO1：NAD(P)H醌脱氢酶1；PA：棕榈酸；PARP：聚（ADP-核糖）聚合酶 1；PRKAA1/2：蛋白激酶 AMP 激活的催化亚基 alpha1/2；RBX1：环盒1；ROS：活性氧；SESN2：sestrin 2；SFA：饱和脂肪酸；siRNA：小干扰RNA；SQSTM1/p62：sequestosome 1；SREBF1：甾醇调节元件结合转录因子1；TBK1：TANK 结合激酶 1；TUNEL：末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记；ULK1：unc-51 像自噬激活激酶。