DJ-1激活Raf/ERK通路促进PC-12细胞自噬成熟

Xue-zhong Li, K. Zhao, Yuansu Zhuang, Xiaopeng Chen, Yi Liu
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引用次数: 0

摘要

Park 7基因编码一种名为DJ-1蛋白的保守蛋白,该蛋白与自噬应激有关,但其机制尚不清楚。因此,有必要探讨DJ-1调节PC-12自噬应激的机制。利用CRISPR/Cas9技术构建DJ-1敲除PC-12细胞系,培养野生型和DJ-1敲除PC-12细胞系,通过MPP+建立氧化应激细胞模型,将其分为野生型对照组(WT)、野生型干预组(WT + MPP+)、DJ-1敲除对照组(KO)和DJ-1敲除干预组(KO + MPP+),通过细胞活力测定、免疫荧光、共聚焦、Western blotting和电子显微镜。结果表明,DJ-1敲除细胞的生长能力低于正常细胞,并且DJ-1敲除细胞对氧化应激更敏感,比野生型细胞更容易受到损伤。暴露于MPP+后,DJ-1蛋白在Cys-106位点发生氧化反应,而DJ-1敲除的PC-12细胞则没有类似的反应。野生型PC-12细胞具有抗氧化DJ-1抗体和抗c - raf磷酸化抗体的共聚焦。活化的DJ-1诱导C-Raf在Ser338位点磷酸化,直接激活C-Raf,随后激活ERK1/2信号通路,拮抗MPP+诱导的神经毒性。缺乏DJ-1,氧化应激不能促进C-Raf的激活。虽然细胞ERK的磷酸化水平也有所升高,但核内pERK的升高并不明显。野生型和DJ-1敲除PC-12细胞在面对氧化应激时均能产生自噬应激,但野生型PC-12细胞产生的自噬溶酶体比例大于DJ-1敲除细胞。PD98059能降低野生型PC-12细胞氧化应激状态下的自噬应激,自噬溶酶体数量也有类似的减少,而索拉非尼对自噬应激有轻微的降低,且自噬溶酶体比例下降更大。因此,我们可以推断,激活的DJ-1直接磷酸化Ser-338位点的C-Raf,然后激活C-Raf,随后激活MEK/ERK通路。DJ-1通过C-Raf/ERK通路促进自噬成熟,从而提高细胞存活率。
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DJ-1 Activation Raf/ERK Pathways Promotes Autophagy Maturation of PC-12 Cells
Park 7 gene encodes a conserved protein called DJ-1 protein, which involves autophagy stress, but the mechanism is unclear. Therefore, it is necessary to explore the mechanism of DJ-1 regulation PC-12 autophagical stress. Using CRISPR/Cas9 technique to construct DJ-1 knockout PC-12 cell lines, we culture wild-type and DJ-1 knockout PC-12 cell lines, establish oxidative stress cell model by MPP+, and divide them into wild-type control group (WT), wild-type intervention group (WT + MPP+), DJ-1 knockout control group (KO) and DJ-1 knockout intervention group (KO + MPP+), and explore the role of DJ-1 in regulating neuronal autophagy stress by cell viability assay, immunofluorescence, confocal, western blotting and electron microscopy. The results show that the growth ability of DJ-1 knockout cells is inferior to that of normal cells, and DJ-1 knockout cells are more sensitive to oxidative stress and more vulnerable to damage than wild-type cells. Exposing to MPP+, DJ-1 proteins undergo oxidative responses at Cys-106 sites, while DJ-1 knockout PC-12 cells do not show similar responses. The wild-type PC-12 cells have the confocal in both anti-oxidant DJ-1 antibody and anti-C-Raf phosphorylation antibody. The activated DJ-1 induces the phosphorylation of C-Raf at Ser338 sites to activate directly C-Raf, and subsequently activates ERK1/2 signaling pathways to antagonize MPP+-induced neurotoxicity. Lack of DJ-1, oxidative stress can not promote C-Raf activation. Although the phosphorylation level of cell ERK is also increased, the increase of intranucleus pERK is not obvious. Wild type and DJ-1 knockout PC-12 cells can produce autophagical stress in the face of oxidative stress, but the proportion of autophagolysosomes produced in wild type PC-12 cells is larger than that in DJ-1 knockout cells. PD98059 can reduce autophagy stress in the state of oxidative stress in wild-type PC-12 cells, and the number of autophagolysosomes is similarly reduced, while sorafenib decreased slightly DJ-1 the autophagical stress, and the proportion of autophagolysosomes decreased more. Therefore, we can infer that activated DJ-1 directly phosphorylates C-Raf at Ser-338 sites, then activating C-Raf, subsequent activation of the MEK/ERK pathway. DJ-1 promotes autophagy maturation through the C-Raf/ERK pathway, thereby improving cell survival.
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