Autophagy deficient keratinocytes display increased DNA damage, senescence and aberrant lipid composition after oxidative stress in vitro and in vivo.
Autophagy deficient keratinocytes display increased DNA damage, senescence and aberrant lipid composition after oxidative stress in vitro and in vivo.
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DOI：
10.1016/j.redox.2016.12.015
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发表时间：
2017-04
期刊：
影响因子：
11.4
通讯作者：
Gruber, Florian
中科院分区：
文献类型：
--
作者：
Song, Xiuzu;Narzt, Marie Sophie;Nagelreiter, Ionela Mariana;Hohensinner, Philipp;Terlecki-Zaniewicz, Lucia;Tschachler, Erwin;Grillari, Johannes;Gruber, Florian
自噬允许细胞对代谢需要和压力进行基本的适应。使用自噬本体降解细胞可以清除氧化还原应激下产生的交联大分子和受损细胞器。这种碎片的积累导致细胞功能障碍，并在老化组织和衰老细胞中观察到。相反，有希望的抗衰老策略旨在抑制mTOR通路，从而激活自噬，以抵消衰老相关的损伤。我们已经在小鼠角质形成细胞（KC）中灭活了自噬相关基因7（Atg 7），这是一种必需的自噬基因，并且在早期的研究中发现，这导致了基线氧化应激增加和氧化紫外线应激后降解交联蛋白的能力降低。为了研究自噬缺陷是否会促进细胞衰老，我们研究了Atg 7缺陷（KO）和Atg 7轴承细胞（WT）如何响应百草枯（PQ）诱导的应激，百草枯是一种常用于诱导细胞衰老的氧化剂药物。与WT相比，Atg 7缺陷型KC显示增加的前列腺素类信号传导和促有丝分裂基因表达特征。暴露于PQ后，WT和KO细胞均显示出炎症和应激相关的转录组学反应。然而，Atg 7缺陷细胞还显示出剧烈的DNA损伤和细胞周期阻滞信号。事实上，DNA断裂和氧化强烈增加，在强调Atg 7缺陷细胞PQ应力后，但也氧化紫外线照射后。损伤相关的磷酸化组蛋白H2 AX（γ H2 AX）灶在细胞核中增加，而核纤层蛋白核纤层蛋白B1的表达强烈下降。类似地，在PQ处理的小鼠尾部皮肤外植体和UVA照射的小鼠尾部皮肤中，我们发现Atg 7缺陷表皮的基底层内γ H2 AX阳性细胞核强烈增加。atg 7缺乏显著影响脂质代谢基因的表达。因此，我们进行了角质形成细胞的脂质分析，这表明细胞脂质代谢的主要失调。我们发现自噬过程中游离脂肪酸的积累，而甘油三酯的水平大大降低。总之，我们的数据表明，在缺乏Atg 7/自噬的情况下，角质形成细胞对内在和环境氧化应激的抵抗力严重受损，并导致DNA损伤、细胞周期停滞和脂质表型紊乱，所有这些都是细胞过早老化的典型表现。角质形成细胞中Atg 7缺失导致的自噬缺陷使其对ROS损伤敏感。ROS应激导致Atg 7 KO中DNA损伤加重和脂质组成扭曲。同样在体内，自噬缺陷细胞倾向于增强DNA损伤。如果没有Autphagy，来自UV和内在来源的ROS可能会造成更高的突变风险。
Autophagy allows cells fundamental adaptations to metabolic needs and to stress. Using autophagic bulk degradation cells can clear crosslinked macromolecules and damaged organelles that arise under redox stress. Accumulation of such debris results in cellular dysfunction and is observed in aged tissue and senescent cells. Conversely, promising anti-aging strategies aim at inhibiting the mTOR pathway and thereby activating autophagy, to counteract aging associated damage. We have inactivated autophagy related 7 (Atg7), an essential autophagy gene, in murine keratinocytes (KC) and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by paraquat (PQ), an oxidant drug commonly used to induce cellular senescence. Atg7 deficient KC displayed increased prostanoid signaling and a pro- mitotic gene expression signature as compared to the WT. After exposure to PQ, both WT and KO cells showed an inflammatory and stress-related transcriptomic response. However, the Atg7 deficient cells additionally showed drastic DNA damage- and cell cycle arrest signaling. Indeed, DNA fragmentation and –oxidation were strongly increased in the stressed Atg7 deficient cells upon PQ stress but also after oxidizing ultraviolet A irradiation. Damage associated phosphorylated histone H2AX (γH2AX) foci were increased in the nuclei, whereas expression of the nuclear lamina protein lamin B1 was strongly decreased. Similarly, in both, PQ treated mouse tail skin explants and in UVA irradiated mouse tail skin, we found a strong increase in γH2AX positive nuclei within the basal layer of Atg7 deficient epidermis. Atg7 deficiency significantly affected expression of lipid metabolic genes. Therefore we performed lipid profiling of keratinocytes which demonstrated a major dysregulation of cellular lipid metabolism. We found accumulation of autophagy agonisitic free fatty acids, whereas triglyceride levels were strongly decreased. Together, our data show that in absence of Atg7/autophagy the resistance of keratinocytes to intrinsic and environmental oxidative stress was severely impaired and resulted in DNA damage, cell cycle arrest and a disturbed lipid phenotype, all typical for premature cell aging. Autphagy deficiency by Atg7 deletion in keratinocytes makes them sensitive to ROS damage. ROS stress causes exacerbated DNA damage and distorted lipid composition in Atg7 KO. Also in vivo the autophagy deficient cells are prone to enhanced DNA damage. Without Autphagy ROS from UV and intrinsic sources may pose higher risk of mutation.
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