Epoxyeicosatrienoic acids (EETs) are potent lipid mediators with well-established effects in vascular tissues. Recent studies indicated an emerging role of these eicosanoids in metabolic diseases and the EET signaling pathway was shown to be involved in hepatic insulin sensitivity. However, compared to vascular tissues, there is only limited knowledge about the underlying signaling pathways in the liver. Therefore, we employed an LC-MS/MS-based time-resolved phosphoproteomics approach to characterize 11,12-EET-mediated signaling events in the liver cell line Hepa 1-6. 11,12-EET treatment resulted in the time-dependent regulation of phosphopeptides involved in processes as yet unknown to be affected by EETs, including RNA processing, splicing and translation regulation. Pathway analysis combined with western blot-based validation revealed enhanced AKT/mTOR/p70S6K signaling as demonstrated by increased acute phosphorylation of AKT (Ser473) and p70S6K (Thr389). In addition, 11,12-EET treatment led to differential regulation of phosphopeptides including important mediators of the DNA damage response and we observed a prolonged induction of the etoposide-induced DNA damage marker γH2AX in response to 11,12-EET. In summary, our findings extend current knowledge of 11,12-EET signaling events and emphasize the importance of the AKT/mTOR/p70S6K pathway in hepatic 11,12-EET signaling. Based on the results presented in this study, we furthermore propose a novel role of EET signaling in the regulation of the DNA damage response.

中文翻译：

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时间分辨的磷酸蛋白质组学分析阐明了肝的11,12-环氧二十碳三烯酸信号通路。

环氧二十碳三烯酸（EET）是有效的脂质介体，在血管组织中具有公认的作用。最近的研究表明，这些类花生酸在代谢性疾病中正在发挥新的作用，并且EET信号通路被证明与肝胰岛素敏感性有关。但是，与血管组织相比，关于肝脏中潜在的信号通路的知识有限。因此，我们采用了基于LC-MS / MS的时间分辨磷酸化蛋白质组学方法来表征肝细胞系Hepa 1-6中的11,12-EET介导的信号转导事件。11,12-EET处理导致涉及EET的过程中涉及到的磷酸肽的时间依赖性调节，这些过程尚不受EET影响，包括RNA加工，剪接和翻译调节。通路分析与基于蛋白质印迹的验证相结合，揭示了增强的AKT / mTOR / p70S6K信号传导，如AKT（Ser473）和p70S6K（Thr389）的急性磷酸化增加所证明。此外，11,12-EET处理导致磷酸肽的差异调节，包括DNA损伤反应的重要介体，我们观察到依托泊苷诱导的对11,12-EET的DNA损伤标记γH2AX的诱导时间延长。总之，我们的发现扩展了对11,12-EET信号事件的当前了解，并强调了AKT / mTOR / p70S6K途径在肝11,12-EET信号中的重要性。基于这项研究中提出的结果，我们进一步提出了EET信号传导在DNA损伤反应调节中的新作用。